Carrier immunoglobulins and uses thereof

ABSTRACT

Disclosed is an isolated immunoglobulin. Also disclosed are pharmaceutical compositions and medicaments comprising the immunoglobulin, isolated nucleic acid encoding it, vectors, host cells, useful in methods of making it. In some embodiments the immunoglobulin comprises one to twenty-four pharmacologically active chemical moieties conjugated thereto, such as a pharmacologically active polypeptide.

This application is a divisional of U.S. application Ser. No.13/825,547, filed Mar. 21, 2013, now allowed, which is a national stageapplication under 35 U.S.C. 371 of International Application No.PCT/US2011/052841, having international filing date of Sep. 22, 2011,which claims the benefit of U.S. Provisional Application No. 61/385,460,filed Sep. 22, 2010. The above-identified applications are each herebyincorporated herein by reference for all purposes.

The instant application is being filed along with a Sequence Listing inelectronic format via EFS-Web. The Sequence Listing is provided as atext file entitled A1536USPCD_st25.txt, created Feb. 19, 2015, which is512,505 bytes in size. The information in the electronic format of theSequence Listing is incorporated herein by reference in its entirety.

Throughout this application various publications are referenced withinparentheses or brackets. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to immunoglobulins to which one or morepharmacologically active chemical moieties can be conjugated forimproved pharmacokinetic characteristics.

2. Discussion of the Related Art

A “carrier” moiety refers to a pharmacologically inactive molecule towhich a pharmacologically active chemical moiety, such as a non-peptideorganic moiety (i.e., “small molecule”) or a polypeptide agent (e.g.,the inventive immmunoglublins), can be covalently conjugated or fused.Effective carriers have been sought to prevent or mitigate in vivodegradation of pharmacologically active moieties by proteolysis or otherin vivo activity-diminishing chemical modifications of thepharmacologically active chemical moiety, or to reduce renal clearance,to enhance in vivo half-life or other pharmacokinetic properties of atherapeutic, such as increasing the rate of absorption, reducingtoxicity or immunogenicity, improving solubility, and/or increasingmanufacturability or storage stability, compared to an unconjugated formof the pharmacologically active moiety.

Examples of such carrier moieties that have been employed in thepharmaceutical industry include polyethylene glycol (see, e.g., Burg etal., Erythropoietin conjugates with polyethylene glycol, WO 01/02017),immunoglobulin Fc domain (see, e.g., Feige et al., Modified peptides astherapeutic agents, U.S. Pat. No. 6,660,843), human serum albumin (see,e.g., Rosen et al., Albumin fusion proteins, U.S. Pat. No. 6,926,898 andUS 2005/0054051; Bridon et al., Protection of endogenous therapeuticpeptides from peptidase activity through conjugation to bloodcomponents, U.S. Pat. No. 6,887,470), transthyretin (see, e.g., Walkeret al., Use of transthyretin peptide/protein fusions to increase theserum half-life of pharmacologically active peptides/proteins, US2003/0195154 A1; 2003/0191056 A1), or thyroxine-binding globulin, or acombination such as immunoglobulin(light chain+heavy chain) and Fcdomain (the heterotrimeric combination a so-called “hemibody”), forexample as described in Sullivan et al., Toxin Peptide TherapeuticAgents, PCT/US2007/022831, published as WO 2008/088422.Pharmacologically active moieties have also been conjugated to a peptideor small molecule that has an affinity for a long half-life serumprotein. (See, e.g., Blaney et al., Method and compositions forincreasing the serum half-life of pharmacologically active agents bybinding to transthyretin-selective ligands, U.S. Pat. No. 5,714,142;Sato et al., Serum albumin binding moieties, US 2003/0069395 A1; Joneset al., Pharmaceutical active conjugates, U.S. Pat. No. 6,342,225).

Fischer et al. described a peptide-immunoglobulin-conjugate, in whichthe immunoglobulin consisted of two heavy chains or two heavy chains andtwo light chains, in which the immunoglobulin was not a functionableimmunoglobulin (Fischer et al., A peptide-immunoglobulin conjugate, WO2007/045463 A1).

The present invention provides immunoglobulins yielding exceptionaluniformity and efficiency of recombinant expression, in vitro stabilityand non-aggregation, resistance to photodegradation and oxidation,non-cross-reactivity with human antigens, and good pharmacokineticproperties.

SUMMARY OF THE INVENTION

The invention relates to immunoglobulins, which are useful as carriermoieties. These immunoglobulins, including antibodies and antibodyfragments, have reliable expression and purification characteristics,resulting in products that are stable and relatively uniform, and haveoutstanding pharmacokinetic (PK) properties in rats and cynomolgousmonkeys. The inventive immunoglobulins have not been detected to bind tohuman proteins, cells or tissues. These immunoglobulins can also be usedfor many purposes, including, but not limited to, quality control oranalytical standards for antibody-based drugs and as controls forbiologically relevant isotype-matched antibodies.

Certain embodiments of the invention include an isolated immunoglobulin,comprising an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region, wherein:

-   -   the heavy chain variable region comprises the amino acid        sequence of SEQ ID NO:323 [VH10] and the light chain variable        region comprises the amino acid sequence of SEQ ID NO:188 [VL4]        or SEQ ID NO:190 [VL5]; or    -   the heavy chain variable region comprises the amino acid        sequence of SEQ ID NO:321 [VH9] and the light chain variable        region comprises the amino acid sequence of SEQ ID NO:188 [VL4]        or SEQ ID NO:190 [VL5]; or    -   the heavy chain variable region comprises the amino acid        sequence of SEQ ID NO:325 [VH11] and the light chain variable        region comprises the amino acid sequence of SEQ ID NO:182 [VL1],        SEQ ID NO:188 [VL4], or SEQ ID NO:190 [VL5].        Examples include antibodies #16435, 16436, 16438, 16439, 16440,        16441, and 16444, disclosed in Table 2C. Typically, the        inventive immunoglobulin at 30 micromolar concentration does not        significantly bind soluble human IL-17R (SEQ ID NO:89) at 30        nanomolar concentration in an aqueous solution incubated under        physiological conditions, e.g., as measured by a surface plasmon        resonance binding assay, as described herein.

Other embodiments of the invention include an isolated immunoglobulin,comprising an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region, wherein:

-   -   the light chain variable region comprises the amino acid        sequence of SEQ ID NO:196 [VL8] and the heavy chain variable        region comprises the amino acid sequence of SEQ ID NO:335        [VH16], SEQ ID NO:349 [VH23], SEQ ID NO:351 [VH24], SEQ ID        NO:353 [VH25], SEQ ID NO:355 [VH26], or SEQ ID NO:359 [VH28]; or    -   the light chain variable region comprises the amino acid        sequence of SEQ ID NO:204 [VL12] and the heavy chain variable        region comprises the amino acid sequence of SEQ ID NO:349 [VH23]        or SEQ ID NO:355 [VH26]; or    -   the light chain variable region comprises the amino acid        sequence of SEQ ID NO:202 [VL11] and the heavy chain variable        region comprises the amino acid sequence of SEQ ID NO:349        [VH23]; or    -   the light chain variable region comprises the amino acid        sequence of SEQ ID NO:192 [VL6] and the heavy chain variable        region comprises the amino acid sequence of SEQ ID NO:357        [VH27], SEQ ID NO:359 [VH28], or SEQ ID NO:369 [VH33]; or    -   the light chain variable region comprises the amino acid        sequence of SEQ ID NO:194 [VL7] and the heavy chain variable        region comprises the amino acid sequence of SEQ ID NO:335        [VH16], SEQ ID NO:349 [VH23], or SEQ ID NO:351 [VH24].        Examples include antibodies #1961, 1962, 1963, 1964, 1965, 1966,        2323, 2324 2330, 4241, 4341, 10182, 10183, 10184, and 10188,        disclosed in Table 2C. Typically, the inventive immunoglobulin        at 10 micromolar concentration does not significantly bind        soluble human TR2 (SEQ ID NO:82) at 10 nanomolar concentration        in an aqueous solution incubated under physiological conditions,        e.g., as measured by a surface plasmon resonance binding assay,        as described herein.

In some embodiments, the immunoglobulin of the present invention is usedas a carrier for pharmacologically active chemical moieties, e.g., smallmolecules, peptides, and/or proteins to enhance their PK properties. Thepharmacologically active moieties can be conjugated, i.e., covalentlybound, to the inventive immunoglobulin by a chemical conjugationreaction, or through recombinant genetic expression, they can be fusedto the immunoglobulin.

The invention also provides materials and methods for producing suchinventive immunoglobulins, including isolated nucleic acids that encodethem, vectors and isolated host cells. Also provided are isolatednucleic acids encoding any of the immunoglobulin heavy and/or lightchain sequences and/or VH and/or VL sequences. In a related embodiment,an expression vector comprising any of the aforementioned nucleic acidsis provided. In still another embodiment, a host cell is providedcomprising any of the aforementioned nucleic acids or expressionvectors.

The inventive immunoglobulin can be used in the manufacture of apharmaceutical composition or medicament. The inventive pharmaceuticalcomposition or medicament comprises the immunoglobulin conjugated with apharmacologically active agent, and a pharmaceutically acceptablediluent, carrier or excipient.

Numerous methods are contemplated in the present invention. For example,a method is provided involving culturing the aforementioned host cellcomprising the expression vector of the invention such that the encodedimmunoglobulin is expressed. Such methods can also comprise the step ofrecovering the immunoglobulin from the host cell culture. In a relatedembodiment, an isolated immunoglobulin produced by the aforementionedmethod is provided.

The foregoing summary is not intended to define every aspect of theinvention, and additional aspects are described in other sections, suchas the Detailed Description of Embodiments. The entire document isintended to be related as a unified disclosure, and it should beunderstood that all combinations of features described herein arecontemplated, even if the combination of features are not found togetherin the same sentence, or paragraph, or section of this document.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations defined by specific paragraphs above. For example,certain aspects of the invention that are described as a genus, and itshould be understood that every member of a genus is, individually, anaspect of the invention. Also, aspects described as a genus or selectinga member of a genus, should be understood to embrace combinations of twoor more members of the genus. Although the applicant(s) invented thefull scope of the invention described herein, the applicants do notintend to claim subject matter described in the prior art work ofothers. Therefore, in the event that statutory prior art within thescope of a claim is brought to the attention of the applicants by aPatent Office or other entity or individual, the applicant(s) reservethe right to exercise amendment rights under applicable patent laws toredefine the subject matter of such a claim to specifically exclude suchstatutory prior art or obvious variations of statutory prior art fromthe scope of such a claim. Variations of the invention defined by suchamended claims also are intended as aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-N shows schematic structures of some embodiments of acomposition of the invention that include one or more units of apharmacologically active toxin peptide analog (squiggle) fused, via anoptional peptidyl linker moiety such as but not limited to L5 or L10described herein, with one or more domains of an immunoglobulin. Theseschematics show a more typical IgG1, although they are intended to applyas well to IgG2s, which will have 4 disulfide bonds in the hinge and adifferent arrangement of the disulfide bond linking the heavy and lightchain, and IgG3s and IgG4s. FIG. 1A represents a monovalentheterodimeric Fc-toxin peptide analog fusion with the toxin peptideanalog fused to the C-terminal end of one of the immunoglobulin Fcdomain monomers. FIG. 1B represents a bivalent homodimeric Fc-toxinpeptide analog fusion, with toxin peptide analogs fused to theC-terminal ends of both of the immunoglobulin Fc domain monomers. FIG.1C represents a monovalent heterodimeric toxin peptide analog-Fc fusionwith the toxin peptide analog fused to the N-terminal end of one of theimmunoglobulin Fc domain monomers. FIG. 1D represents a bivalenthomodimeric toxin peptide analog-Fc fusion, with toxin peptide analogsfused to the N-terminal ends of both of the immunoglobulin Fc domainmonomers. FIG. 1E represents a monovalent heterotrimeric Fc-toxinpeptide analog/Ab comprising an immunoglobulin heavy chain(HC)+immunoglobulin light chain (LC)+an immunoglobulin Fc monomer with atoxin peptide analog fused to its C-terminal end. FIG. 1F represents amonovalent heterotetrameric (HT) antibody HC-toxin peptide analogfusion, with a toxin peptide analog fused to the C-terminal end of oneof the HC monomers. FIG. 1G represents a bivalent HT antibody AbHC-toxin peptide analog fusion having toxin peptide analogs on theC-terminal ends of both HC monomers. FIG. 1H represents a monovalent HTtoxin peptide analog-LC Ab, with the toxin peptide analog fused to theN-terminal end of one of the LC monomers. FIG. 1I represents amonovalent HT toxin peptide analog-HC Ab, with the toxin peptide analogfused to the N-terminal end of one of the HC monomers. FIG. 1Jrepresents a monovalent HT Ab LC-toxin peptide analog fusion (i.e.,LC-toxin peptide analog fusion+LC+2(HC)), with the toxin peptide analogfused to the C-terminal end of one of the LC monomers. FIG. 1Krepresents a bivalent HT Ab LC-toxin peptide analog fusion (i.e.,2(LC-toxin peptide analog fusion)+2(HC)), with toxin peptide analogsfused to the C-terminal end of both of the LC monomers. FIG. 1Lrepresents a trivalent HT Ab LC-toxin peptide analog/HC-toxin peptideanalog (i.e., 2(LC-toxin peptide analog fusion)+HC-toxin peptide analogfusion+HC), with the toxin peptide analogs fused to the C-terminal endsof both of the LC monomers and one of the HC monomers. FIG. 1Mrepresents a bivalent antibody with a toxin peptide analog moietyinserted into an internal loop of the immunoglobulin Fc domain of eachHC monomer. FIG. 1N represents a monovalent antibody with a toxinpeptide analog moiety inserted into an internal loop of theimmunoglobulin Fc domain of one of the HC monomers. Dimers or trimerswill form spontaneously in certain host cells upon expression of adeoxyribonucleic acid (DNA) construct encoding a single chain. In otherhost cells, the cells can be placed in conditions favoring formation ofdimers/trimers or the dimers/trimers can be formed in vitro. If morethan one HC monomer, LC monomer, or immunoglobulin Fc domain monomer ispart of a single embodiment, the individual monomers can be, if desired,identical or different from each other.

FIGS. 2A-B shows non-binding to IL17R by antibody embodiments of thepresent invention. Antibody 16429 was immobilized to a CM5 sensor chip,and 10 nM of IL-17R in the absence of antibody was used to establish the100% binding signal of IL-17 that is free of antibody binding insolution. In FIG. 2A, 10 nM, 100 nM and 1000 nM of indicated antibodysamples were incubated with the 10 nM IL-17R to determine antibodybinding in solution. In FIG. 2B, 30,000 nM of the antibody samples wereincubated with 30 nM IL-17R to determine antibody binding in solution.In FIGS. 2A-B, The decreased binding signal of IL-17R after the antibodyincubation indicates the binding of the antibody to IL-17R in solution.

FIG. 3 shows relative production of GRO-α by human foreskin fibroblasts,which were incubated with 5 ng/ml IL-17 and 0.1 μM, 1 μM, and 10 μM ofthe indicated antibody samples. The conditioned cell medium was thenassessed for GRO-αlevels using a GRO-α sandwich ELISA.

FIG. 4A shows representative elutions from two size exclusion columns inseries (TSK-GEL G3000SWXL, 5 mm particle size, 7.8×300 mm,TosohBioscience, 08541) with a 100 mM sodium phosphate, 250 mM NaCl atpH 6.8 mobile phase flowed at 0.5 mL/min., of antibodies (top to bottompanels): 16435, 16436, 16439, 16440, 16441 and 16444.

FIG. 4B shows a zoomed analysis of the size exclusion analysis shown inFIG. 4A above, of antibodies (top to bottom panels): 16435, 16436,16439, 16440, 16441 and 16444.

FIG. 5 shows non-reducing analysis of 2 μg of antibodies 16435, 16436,16437, 16438, 16439, 16440, 16429, 16430, 16433, 16434, 16441 and 16444on 1.0 mm Tris-glycine 4-20% SDS-PAGEs (Novex) developed at 220V usingnon-reducing loading buffer and staining with QuickBlue (BostonBiologicals). Molecular weight markers are Novex SeeBlue® pre-stainedstandards.

FIG. 6 shows reducing analysis of 2 μg of antibodies 16435, 16436,16437, 16438, 16439, 16440, 16429, 16430, 16433, 16434, 16441 and 16444on 1.0 mm Tris-glycine 4-20% SDS-PAGEs (Novex) developed at 220V usingnon-reducing loading buffer and staining with QuickBlue (BostonBiologicals). Molecular weight markers are Novex SeeBlue® pre-stainedstandards.

FIGS. 7A-B shows titers for antibodies 16435 and 16444, respectively.Expressing pools were created by transfecting CHO DHFR(−) host cellswith corresponding HC and LC expression plasmid. Small scale (5-mL; FIG.7A) expression runs were conducted using a 6-day front-loaded process inCD 6-D assay media, while the large scale (3-L; FIG. 7B) runs werecompleted using an 11-day fed-batch process with peptone medium. Titerlevels were measured using a protein A HPLC based assay.

FIGS. 8A-B shows chomatograms of antibodies 16435 (FIG. 8A) and 16444(FIG. 8B) eluted from a SP-HP sepharose column (GE Life Sciences) usinga 20 column volume gradient to 50% S-Buffer B (20 mM acetic acid, 1 MNaCl, pH 5.0) at 7° C., measuring the absorbance at 300 nm.

FIGS. 9A-B shows analysis of the 16435 (FIG. 9A) and 16444 (FIG. 9B)antibodies on 1.0 mm Tris-glycine 4-20% SDS-PAGEs (Novex) developed at220V staining with QuickBlue (Boston Biologicals). The lanes marked “NR”contained non-reducing sample buffer, while those in lanes marked “Red.”contained reducing sample buffer.

FIG. 10 shows zoomed size exclusion analysis, using two size exclusioncolumns in series (TSK-GEL G3000SWXL, 5 mm particle size, 7.8×300 mm,TosohBioscience, 08541) with a 100 mM sodium phosphate, 250 mM NaCl atpH 6.8 mobile phase flowed at 0.5 mL/min., of antibodies: 16435 and16444.

FIG. 11 shows an analysis of antibodies: an IgG2 monoclonal antibodycomparator, 16444, and 16435, by DSC using a MicroCal VP-DSC where thesamples were heated from 20° C. to 95° C. at a rate of 1° C. per minute.The protein concentration was 0.5 mg/ml in 10 mM sodium acetate, 9%sucrose, pH 5.0.

FIGS. 12A-D shows an analysis of 16435 (FIGS. 12A-B) and 16444 (FIGS.12C-D) antibodies by reducing (FIG. 12A and FIG. 12C) and non-reducing(FIG. 12B and FIG. 12D) CE-SDS with detection of absorbance at 220 nm. Abare-fused silica capillary 50 μm×30.2 cm was used for the separationanalysis.

FIG. 13 shows size exclusion analysis of antibodies 16435 and 16444after 3 days at room temperature covered in aluminum foil (“dark”) orexposed to fluorescent light (“light”), eluted from two size exclusioncolumns in series (TSK-GEL G3000SWXL, 5 mm particle size, 7.8×300 mm,TosohBioscience, 08541) with a 100 mM sodium phosphate, 250 mM NaCl atpH 6.8 mobile phase flowed at 0.5 mL/min.

FIGS. 14A-D shows HIC analysis of the 16435 (FIG. 14A and FIG. 14C) and16444 (FIG. 14B and FIG. 14D) antibodies, after 3 days at roomtemperature covered in aluminum foil (“dark”, FIGS. 14A-B) or exposed tofluorescent light (“light”, FIG. 14C-D), using two Dionex ProPac HIC-10columns in series with mobile phase A being 1 M ammonium sulfate, 20 mMsodium acetate, pH 5.0 and mobile phase B being 20 mM sodium acetate, 5%acetonitrile, pH 5.0. Samples were eluted at 0.8 ml/min with a 0-100%linear gradient over 50 minutes, measuring the absorbance at 220 nm.

FIG. 15 shows binding of antibody to TRAIL (huTR2). Antibody 16449 wasimmobilized to a CM5 sensor chip, and 1 nM of TRAIL in the absence ofantibody was used to establish the 100% binding signal of TRAIL that isfree of antibody binding in solution. To determine antibody binding insolution, 7 pM to 10 nM of the antibody samples were incubated with the1 nM TRAIL. The decreased binding signal of TRAIL after the antibodyincubation indicates the binding of the antibody to TRAIL in solution.

FIG. 16 shows non-binding to TRAIL (huTR2) by antibody embodiments ofthe present invention. Antibody 16449 was immobilized to a CM5 sensorchip, and 10 nM of TRAIL in the absence of antibody was used toestablish the 100% binding signal of TRAIL that is free of antibodybinding in solution. To determine antibody binding in solution, 50 and1000 nM of the antibody samples were incubated with the 10 nM TRAIL. Thedecreased binding signal of TRAIL after the antibody incubationindicates the binding of the antibody to TRAIL in solution.

FIG. 17 shows non-binding to TRAIL (huTR2) by antibody embodiments ofthe present invention. Antibody 16449 was immobilized to a CM5 sensorchip, and 10 nM of TRAIL in the absence of antibody was used toestablish the 100% binding signal of TRAIL that is free of antibodybinding in solution. To determine antibody binding in solution, 1000 nMof the antibody samples were incubated with the 10 nM TRAIL. Thedecreased binding signal of TRAIL after the antibody incubationindicates the binding of the antibody to TRAIL in solution.

FIG. 18 shows non-binding to TRAIL (huTR2) by antibody embodiments ofthe present invention, listed on the y-axis. Antibody 16449 wasimmobilized to a CM5 sensor chip, and 10 nM of TRAIL in the absence ofantibody was used to establish the 100% binding signal of TRAIL that isfree of antibody binding in solution. To determine antibody binding insolution, 1000 and 10000 nM of the antibody samples were incubated withthe 10 nM TRAIL. The decreased binding signal of TRAIL after theantibody incubation indicates the binding of the antibody to TRAIL insolution.

FIG. 19 shows non-binding to TRAIL (huTR2) by antibody embodiments ofthe present invention, listed on the x-axis. Antibody 16449 wasimmobilized to a CM5 sensor chip, and 10 nM of TRAIL in the absence ofantibody was used to establish the 100% binding signal of TRAIL that isfree of antibody binding in solution. To determine antibody binding insolution, 50000 nM of the antibody samples were incubated with the 10 nMTRAIL. The decreased binding signal of TRAIL after the antibodyincubation indicates the binding of the antibody to TRAIL in solution.

FIGS. 20A-B shows non-binding to TRAIL (huTR2) by antibody embodimentsof the present invention, listed on the x-axis. Antibody 16449 wasimmobilized to a CM5 sensor chip, and 10 nM of TRAIL in the absence ofantibody was used to establish the 100% binding signal of TRAIL that isfree of antibody binding in solution. To determine antibody binding insolution, 1000, 10000 and 50000 nM of the antibody samples wereincubated with the 10 nM TRAIL. The decreased binding signal of TRAILafter the antibody incubation indicates the binding of the antibody toTRAIL in solution.

FIG. 20C shows results from an in vitro cell-based TRAIL activity assay.Samples of antibodies 4241 and 4341 were compared with positive controlIgG1 anti-TR2 mAb 16449. The prepared antibody samples were added toTRAIL-sensitive human ascites colorectal adenocarcinoma cell lineColo205. The detection of TRAIL-mediated caspase-3 activation bymeasuring an increase in relative luminescence was used as a positivemarker for apoptosis. Antibodies 4241 and 4341 failed to activatecaspase-3, unlike positive control antibody 16449.

FIG. 21A shows non-reducing analysis of 2 μg of antibodies 1870 [aka16451], 16449, 16450, 10185, 10184, 4341, 10183 and 10182 on 1.0 mmTris-glycine 4-20% SDS-PAGEs (Novex) developed at 220V usingnon-reducing loading buffer and staining with QuickBlue (BostonBiologicals). Molecular weight markers are Novex SeeBlue pre-stainedstandards. Molecular weight markers are Novex SeeBlue® pre-stainedstandards.

FIG. 21B shows reducing analysis of 2 μg of antibodies 1870 [aka16451],16449, 16450, 10185, 10184, 4341, 10183 and 10182 on 1.0 mm Tris-glycine4-20% SDS-PAGEs (Novex) developed at 220V using non-reducing loadingbuffer and staining with QuickBlue (Boston Biologicals). Molecularweight markers are Novex SeeBlue® pre-stained standards.

FIGS. 22A-F shows size exclusion chromatography on 50 μg of antibodies4241 (FIG. 22A), 4341 (FIG. 22B), 10182 (FIG. 22C), 10183 (FIG. 22D),10184 (FIG. 22E), and 10185 (FIG. 22F), injected on to a PhenomenexBioSep SEC-3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, andpH 6.9 at 1 mL/min, measuring the absorbance at 280 nm.

FIGS. 23A-B shows titers for antibodies 4241 and 4341, respectively.Expressing pools were created by transfecting CHO DHFR(−) host cellswith corresponding HC and LC expression plasmid. Small scale (5-mL; FIG.23A) expression runs were conducted using a 6-day front-loaded processin CD 6-D assay media, while the large scale (3-L; FIG. 23B) runs werecompleted using an 11-day fed-batch process with peptone medium. Titerlevels were measured using a protein A HPLC based assay.

FIGS. 24A-B shows reducing analysis of the in process samples forantibodies 4241 (FIG. 24A) and 4341 (FIG. 24B) on 1.0 mm Tris-glycine4-20% SDS-PAGEs (Novex) developed at 220V using non-reducing loadingbuffer and staining with QuickBlue (Boston Biologicals).

FIG. 25 shows an overlay of the chomatograms of antibodies 4341 and 4241on an SP-HP sepharose column (GE Life Sciences) eluted using a 20 columnvolume gradient to 50% S-Buffer B (20 mM acetic acid, 1 M NaCl, pH 5.0)at 7° C. observing the absorbance at 300 nm.

FIGS. 26A-B shows analysis of the 4241 (FIG. 26A) and 4341 (FIG. 26B)antibodies on 1.0 mm Tris-glycine 4-20% SDS-PAGEs (Novex) developed at220V staining with QuickBlue (Boston Biologicals). The lanes marked “NR”contained non-reducing sample buffer, while those in lanes marked “Red.”contained reducing sample buffer.

FIGS. 27A-B shows full scale (FIG. 27A) and zoomed (FIG. 27B) analysis,using two size exclusion columns (TSK-GEL G3000SWXL, 5 mm particle size,7.8×300 mm, TosohBioscience, 08541) in series with a 100 mM sodiumphosphate, 250 mM NaCl at pH 6.8 mobile phase flowed at 0.5 mL/min., ofantibodies: 4241 (upper panels) and 4341 (lower panels).

FIG. 28 shows an analysis of antibodies 4341 and 4241 by DSC using aMicroCal VP-DSC where the samples were heated from 20° C. to 95° C. at arate of 1° C. per minute. The protein concentration was 0.5 mg/ml in 10mM sodium acetate, 9% sucrose, pH 5.0.

FIGS. 29A-D shows an analysis of 4241 (FIGS. 29A-B) and 4341 (FIGS.29C-D) antibodies by reducing (FIG. 29A and FIG. 29C) and non-reducing(FIG. 29B and FIG. 29D) CE-SDS with detection of absorbance at 220 nm. Abare-fused silica capillary 50 μm×30.2 cm was used for the separationanalysis.

FIG. 30 shows analysis of the 4241 (upper panel) and 4341 (lower panel)antibodies using ion exchange HPLC (SP-5PW, 10 μm particle, 7.5 mmID×7.5 cm, TosohBioscience, 08541) using 20 mM acetic acid, pH 5.0 asbuffer A and 20 mM acetic acid, 1 M NaCl, pH 5.0 as buffer B flowed at 1mL/min with an 80 minute linear gradient from 0-40% buffer B.

FIGS. 31A-B shows HIC analysis of the 4241 (FIG. 31A) and 4341 (FIG.31B) antibodies, before and after light exposure, using two DionexProPac HIC-10 columns in series with mobile phase A being 1 M ammoniumsulfate, 20 mM sodium acetate, pH 5.0 and mobile phase B being 20 mMsodium acetate, 5% acetonitrile, pH 5.0. Samples were eluted at 0.8ml/min with a 0-100% linear gradient over 50 minutes observing theabsorbance at 220 nm.

FIG. 32 shows representative pharmacokinetic profiles of the 16435,16444, 4241, and 4341 antibodies, as determined in adult Sprague-Dawleyrats (8-12 weeks old) by injecting 5 mg/kg subcutaneously and collectingblood at 0, 0.25, 1, 4, 24, 48, 72, 96, 168, 336, 504, 672, 840 and 1008hours post-dose from the lateral tail vein. Serum concentrations werethen determined using an anti-human Fc based ELISA.

FIG. 33 shows representative pharmacokinetic profiles of the 16435antibody, as determined in male cynomolgus monkeys using a single IVdose at either 1 mg/kg or 10 mg/kg. Serum samples were collectedpre-dose and at 0.25, 0.5, 1, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168,192, 216, 240, 264, 288, 312, 360, 408, 456, 504, 552, 600, 648 and 672hours after administration. Samples were assayed for 16435 antibodylevels using an anti-IgG sandwich ELISA.

FIG. 34 shows a schematic structural representation of one embodiment ofa composition of the invention that includes one unit of apharmacologically active toxin peptide analog (squiggle) fused, via anoptional peptidyl linker moiety with one immunoglobulin.

FIG. 35 shows a Coomassie brilliant blue-stained Tris-glycine 4-20%SDS-PAGE of final monovalent 16435 IgG2-L10-Shk[1-35, Q16K] products.Products were isolated from four different expression pools. Lanes 1-10were loaded as follows: Novex Mark12 wide range protein standards (10μl), 2 μg pool 1 product non-reduced, 2 μg pool 2 product non-reduced, 2μg pool 3 product non-reduced, 2 μg pool 4 product non-reduced, NovexMark12 wide range protein standards (10 μl), 2 μg pool 1 productreduced, 2 μg pool 2 product reduced, 2 μg pool 3 product reduced, 2 μgpool 4 product reduced.

FIGS. 36A-D shows size exclusion chromatography on 30 μg of the finalpool 1, 2, 3 & 4 of the 3742 product injected onto a Phenomenex BioSepSEC-3000 column (7.8×300 mm) equilibrated in 50 mM NaH2PO4, 250 mM NaCl,pH 6.9 at 1 ml/min, measuring the absorbance at 280 nm.

FIGS. 37A-D shows reduced light chain LC-MS analysis of the final 3742samples. The product was chromatographed through a Waters MassPREP microdesalting column using a Waters ACQUITY UPLC system. The column was setat 80° C. and the protein eluted using a linear gradient of increasingacetonitrile concentration in 0.1% formic acid. The column effluent wasdirected into a Waters LCT Premier ESI-TOF mass spectrometer for massanalysis. The instrument was run in the positive V mode. The capillaryvoltage was set at 3,200 V and the cone voltage at 80 V. The massspectrum was acquired from 800 to 3000 m/z and deconvoluted using theMaxEnt1 software provided by the instrument manufacturer.

FIGS. 38A-D shows reduced heavy chain LC-MS analysis of the final 3742samples. The product was chromatographed through a Waters MassPREP microdesalting column using a Waters ACQUITY UPLC system. The column was setat 80° C. and the protein eluted using a linear gradient of increasingacetonitrile concentration in 0.1% formic acid. The column effluent wasdirected into a Waters LCT Premier ESI-TOF mass spectrometer for massanalysis. The instrument was run in the positive V mode. The capillaryvoltage was set at 3,200 V and the cone voltage at 80 V. The massspectrum was acquired from 800 to 3000 m/z and deconvoluted using theMaxEnt1 software provided by the instrument manufacturer.

FIG. 39A shows non-reducing analysis of the conditioned media ofantibody fusions 10162, 10163 and 10164, along with the conditionedmedia from a mock transfection, on 1.0 mm Tris-glycine 4-20% SDS-PAGEs(Novex) developed at 220V using non-reducing loading buffer and stainingwith QuickBlue (Boston Biologicals). Molecular weight markers areindicated in kDa.

FIG. 39B shows a Coomassie brilliant blue stained Tris-glycine 4-20%SDS-PAGE of final 10162, 10163 & 10164 products. In lanes 1 & 5, NovexMark 12 standards were loaded. For lanes 2-4 (non-reducing) and 6-8(reducing), 2 μg of product was loaded.

FIGS. 40A-C shows size exclusion chromatography on 50 μg of fusionantibodies 10162 (FIG. 40A), 10163 (FIG. 40B), and 10164 (FIG. 40C)injected on to a Phenomenex BioSep SEC-3000 column (7.8×300 mm) in 50 mMNaH₂PO₄, 250 mM NaCl, and pH 6.9 at 1 mL/min measuring the absorbance at280 nm.

FIGS. 41A-C shows reduced light chain LC-MS analysis of the final4341-ShK(1-35, Q16K) (FIG. 41A), 4341-FGF21 (FIG. 41B), and 16435-FGF21(FIG. 41C) samples. The product was chromatographed through a WatersMassPREP micro desalting column using a Waters ACQUITY UPLC system. Thecolumn was set at 80° C. and the protein eluted using a linear gradientof increasing acetonitrile concentration in 0.1% formic acid. The columneffluent was directed into a Waters LCT Premier ESI-TOF massspectrometer for mass analysis. The instrument was run in the positive Vmode. The capillary voltage was set at 3,200 V and the cone voltage at80 V. The mass spectrum was acquired from 800 to 3000 m/z anddeconvoluted using the MaxEnt1 software provided by the instrumentmanufacturer.

FIGS. 42A-C shows reduced heavy chain LC-MS analysis of the final4341-ShK (1-35, Q16K) (FIG. 42A), 4341-FGF21 (FIG. 42B), and 16435-FGF21(FIG. 42C) samples. The product was chromatographed through a WatersMassPREP micro desalting column using a Waters ACQUITY UPLC system. Thecolumn was set at 80° C. and the protein eluted using a linear gradientof increasing acetonitrile concentration in 0.1% formic acid. The columneffluent was directed into a Waters LCT Premier ESI-TOF massspectrometer for mass analysis. The instrument was run in the positive Vmode. The capillary voltage was set at 3,200 V and the cone voltage at80 V. The mass spectrum was acquired from 800 to 3000 m/z anddeconvoluted using the MaxEnt1 software provided by the instrumentmanufacturer.

FIG. 43 shows representative PK profiles of antibodies 16435 and 4341(both at 5 mg/kg dose) in SD rats.

FIG. 44 shows representative PK profiles for sequential doses (5 mg/kg)of antibodies 16435 or 4341 in cynomolgus monkeys.

DETAILED DESCRIPTION OF EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

DEFINITIONS

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Thus, as usedin this specification and the appended claims, the singular forms “a”,“an” and “the” include plural referents unless the context clearlyindicates otherwise. For example, reference to “a protein” includes aplurality of proteins; reference to “a cell” includes populations of aplurality of cells.

“Polypeptide” and “protein” are used interchangeably herein and includea molecular chain of two or more amino acids linked covalently throughpeptide bonds. The terms do not refer to a specific length of theproduct. Thus, “peptides,” and “oligopeptides,” are included within thedefinition of polypeptide. The terms include post-translationalmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. In addition, proteinfragments, analogs, mutated or variant proteins, fusion proteins and thelike are included within the meaning of polypeptide. The terms alsoinclude molecules in which one or more amino acid analogs ornon-canonical or unnatural amino acids are included as can be expressedrecombinantly using known protein engineering techniques. In addition,fusion proteins can be derivatized as described herein by well-knownorganic chemistry techniques.

The term “isolated protein” referred means that a subject protein (1) isfree of at least some other proteins with which it would normally befound in nature, (2) is essentially free of other proteins from the samesource, e.g., from the same species, (3) is expressed recombinantly by acell of a heterologous species or kind, (4) has been separated from atleast about 50 percent of polynucleotides, lipids, carbohydrates, orother materials with which it is associated in nature, (5) is operablyassociated (by covalent or noncovalent interaction) with a polypeptidewith which it is not associated in nature, and/or (6) does not occur innature. Typically, an “isolated protein” constitutes at least about 5%,at least about 10%, at least about 25%, or at least about 50% of a givensample. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, orany combination thereof may encode such an isolated protein. Preferably,the isolated protein is substantially free from proteins or polypeptidesor other contaminants that are found in its natural environment thatwould interfere with its therapeutic, diagnostic, prophylactic, researchor other use.

A “variant” of a polypeptide (e.g., an immunoglobulin, or an antibody)comprises an amino acid sequence wherein one or more amino acid residuesare inserted into, deleted from and/or substituted into the amino acidsequence relative to another polypeptide sequence. Variants includefusion proteins.

The term “fusion protein” indicates that the protein includespolypeptide components derived from more than one parental protein orpolypeptide. Typically, a fusion protein is expressed from a fusion genein which a nucleotide sequence encoding a polypeptide sequence from oneprotein is appended in frame with, and optionally separated by a linkerfrom, a nucleotide sequence encoding a polypeptide sequence from adifferent protein. The fusion gene can then be expressed by arecombinant host cell as a single protein.

A “secreted” protein refers to those proteins capable of being directedto the ER, secretory vesicles, or the extracellular space as a result ofa secretory signal peptide sequence, as well as those proteins releasedinto the extracellular space without necessarily containing a signalsequence. If the secreted protein is released into the extracellularspace, the secreted protein can undergo extracellular processing toproduce a “mature” protein. Release into the extracellular space canoccur by many mechanisms, including exocytosis and proteolytic cleavage.In some other embodiments of the inventive composition, the toxinpeptide analog can be synthesized by the host cell as a secretedprotein, which can then be further purified from the extracellular spaceand/or medium.

As used herein “soluble” when in reference to a protein produced byrecombinant DNA technology in a host cell is a protein that exists inaqueous solution; if the protein contains a twin-arginine signal aminoacid sequence the soluble protein is exported to the periplasmic spacein gram negative bacterial hosts, or is secreted into the culture mediumby eukaryotic host cells capable of secretion, or by bacterial hostpossessing the appropriate genes (e.g., the kil gene). Thus, a solubleprotein is a protein which is not found in an inclusion body inside thehost cell. Alternatively, depending on the context, a soluble protein isa protein which is not found integrated in cellular membranes; incontrast, an insoluble protein is one which exists in denatured forminside cytoplasmic granules (called an inclusion body) in the host cell,or again depending on the context, an insoluble protein is one which ispresent in cell membranes, including but not limited to, cytoplasmicmembranes, mitochondrial membranes, chloroplast membranes, endoplasmicreticulum membranes, etc.

“Soluble human IL-17R” is a polypeptide (huIL-17R-FpH) having thefollowing amino acid sequence:

SEQ ID NO: 89 LRLLDHRALVCSQPGLNCTVKNSTCLDDSWIHPRNLTPSSPKDLQIQLHFAHTQQGDLFPVAHIEWTLQTDASILYLEGAELSVLQLNTNERLCVRFEFLSKLRHHHRRWRFTFSHFVVDPDQEYEVTVHHLPKPIPDGDPNHQSKNFLVPDCEHARMKVTTPCMSSGSLWDPNITVETLEAHOLRVSFTLWNESTHYQILLTSFPHMENHSCFEHMHHIPAPRPEEFHQRSNVTLTLRNLKGCCRHQVQIQPFFSSCLNDCLRHSATVSCPEMPDTPEPIPDYMPLWEPRSGSSDYKDD DDKGSSHHHHHH//.

“Soluble human TR2” is a fusion polypeptide (huTR2 long-huFc (IgG1), inmonomeric or dimeric form, having the following amino acid sequence:

SEQ ID NO: 82 MEQRGQNAPAASGARKRHGPGPREARGARPGPRVPKTLVLVVAAVLLLVSAESALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEAPAVEETVTSSPGTPASPCSLSGVDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//.

“Under physiological conditions” with respect to incubating buffers andimmunoglobulins, or other binding assay reagents means incubation underconditions of temperature, pH, and ionic strength, that permit abiochemical reaction, such as a non-covalent binding reaction, to occur.Typically, the temperature is at room or ambient temperature up to about37° C. and at pH 6.5-7.5.

The term “recombinant” indicates that the material (e.g., a nucleic acidor a polypeptide) has been artificially or synthetically (i.e.,non-naturally) altered by human intervention. The alteration can beperformed on the material within, or removed from, its naturalenvironment or state. For example, a “recombinant nucleic acid” is onethat is made by recombining nucleic acids, e.g., during cloning, DNAshuffling or other well known molecular biological procedures. Examplesof such molecular biological procedures are found in Maniatis et al.,Molecular Cloning. A Laboratory Manual. Cold Spring Harbour Laboratory,Cold Spring Harbour, N.Y (1982). A “recombinant DNA molecule,” iscomprised of segments of DNA joined together by means of such molecularbiological techniques. The term “recombinant protein” or “recombinantpolypeptide” as used herein refers to a protein molecule which isexpressed using a recombinant DNA molecule. A “recombinant host cell” isa cell that contains and/or expresses a recombinant nucleic acid.

The term “polynucleotide” or “nucleic acid” includes bothsingle-stranded and double-stranded nucleotide polymers containing twoor more nucleotide residues. The nucleotide residues comprising thepolynucleotide can be ribonucleotides or deoxyribonucleotides or amodified form of either type of nucleotide. Said modifications includebase modifications such as bromouridine and inosine derivatives, ribosemodifications such as 2′,3′-dideoxyribose, and internucleotide linkagemodifications such as phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoraniladate and phosphoroamidate.

The term “oligonucleotide” means a polynucleotide comprising 200 orfewer nucleotide residues. In some embodiments, oligonucleotides are 10to 60 bases in length. In other embodiments, oligonucleotides are 12,13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in length.Oligonucleotides may be single stranded or double stranded, e.g., foruse in the construction of a mutant gene. Oligonucleotides may be senseor antisense oligonucleotides. An oligonucleotide can include a label,including a radiolabel, a fluorescent label, a hapten or an antigeniclabel, for detection assays. Oligonucleotides may be used, for example,as PCR primers, cloning primers or hybridization probes.

A “polynucleotide sequence” or “nucleotide sequence” or “nucleic acidsequence,” as used interchangeably herein, is the primary sequence ofnucleotide residues in a polynucleotide, including of anoligonucleotide, a DNA, and RNA, a nucleic acid, or a character stringrepresenting the primary sequence of nucleotide residues, depending oncontext. From any specified polynucleotide sequence, either the givennucleic acid or the complementary polynucleotide sequence can bedetermined. Included are DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand. Unless specified otherwise, the left-hand end of anysingle-stranded polynucleotide sequence discussed herein is the 5′ end;the left-hand direction of double-stranded polynucleotide sequences isreferred to as the 5′ direction. The direction of 5′ to 3′ addition ofnascent RNA transcripts is referred to as the transcription direction;sequence regions on the DNA strand having the same sequence as the RNAtranscript that are 5′ to the 5′ end of the RNA transcript are referredto as “upstream sequences;” sequence regions on the DNA strand havingthe same sequence as the RNA transcript that are 3′ to the 3′ end of theRNA transcript are referred to as “downstream sequences.”

As used herein, an “isolated nucleic acid molecule” or “isolated nucleicacid sequence” is a nucleic acid molecule that is either (1) identifiedand separated from at least one contaminant nucleic acid molecule withwhich it is ordinarily associated in the natural source of the nucleicacid or (2) cloned, amplified, tagged, or otherwise distinguished frombackground nucleic acids such that the sequence of the nucleic acid ofinterest can be determined. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. However, anisolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the immunoglobulin (e.g.,antibody) where, for example, the nucleic acid molecule is in achromosomal location different from that of natural cells.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of ribonucleotidesalong the mRNA chain, and also determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for the RNAsequence and for the amino acid sequence.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. Genes typically include coding sequencesand/or the regulatory sequences required for expression of such codingsequences. The term “gene” applies to a specific genomic or recombinantsequence, as well as to a cDNA or mRNA encoded by that sequence. A“fusion gene” contains a coding region that encodes a toxin peptideanalog. Genes also include non-expressed nucleic acid segments that, forexample, form recognition sequences for other proteins. Non-expressedregulatory sequences including transcriptional control elements to whichregulatory proteins, such as transcription factors, bind, resulting intranscription of adjacent or nearby sequences.

“Expression of a gene” or “expression of a nucleic acid” meanstranscription of DNA into RNA (optionally including modification of theRNA, e.g., splicing), translation of RNA into a polypeptide (possiblyincluding subsequent post-translational modification of thepolypeptide), or both transcription and translation, as indicated by thecontext.

As used herein the term “coding region” or “coding sequence” when usedin reference to a structural gene refers to the nucleotide sequenceswhich encode the amino acids found in the nascent polypeptide as aresult of translation of an mRNA molecule. The coding region is bounded,in eukaryotes, on the 5′ side by the nucleotide triplet “ATG” whichencodes the initiator methionine and on the 3′ side by one of the threetriplets which specify stop codons (i.e., TAA, TAG, TGA).

The term “control sequence” or “control signal” refers to apolynucleotide sequence that can, in a particular host cell, affect theexpression and processing of coding sequences to which it is ligated.The nature of such control sequences may depend upon the host organism.In particular embodiments, control sequences for prokaryotes may includea promoter, a ribosomal binding site, and a transcription terminationsequence. Control sequences for eukaryotes may include promoterscomprising one or a plurality of recognition sites for transcriptionfactors, transcription enhancer sequences or elements, polyadenylationsites, and transcription termination sequences. Control sequences caninclude leader sequences and/or fusion partner sequences. Promoters andenhancers consist of short arrays of DNA that interact specifically withcellular proteins involved in transcription (Maniatis, et al., Science236:1237 (1987)). Promoter and enhancer elements have been isolated froma variety of eukaryotic sources including genes in yeast, insect andmammalian cells and viruses (analogous control elements, i.e.,promoters, are also found in prokaryotes). The selection of a particularpromoter and enhancer depends on what cell type is to be used to expressthe protein of interest. Some eukaryotic promoters and enhancers have abroad host range while others are functional in a limited subset of celltypes (for review see Voss, et al., Trends Biochem. Sci., 11:287 (1986)and Maniatis, et al., Science 236:1237 (1987)).

The term “vector” means any molecule or entity (e.g., nucleic acid,plasmid, bacteriophage or virus) used to transfer protein codinginformation into a host cell.

The term “expression vector” or “expression construct” as used hereinrefers to a recombinant DNA molecule containing a desired codingsequence and appropriate nucleic acid control sequences necessary forthe expression of the operably linked coding sequence in a particularhost cell. An expression vector can include, but is not limited to,sequences that affect or control transcription, translation, and, ifintrons are present, affect RNA splicing of a coding region operablylinked thereto. Nucleic acid sequences necessary for expression inprokaryotes include a promoter, optionally an operator sequence, aribosome binding site and possibly other sequences. Eukaryotic cells areknown to utilize promoters, enhancers, and termination andpolyadenylation signals. A secretory signal peptide sequence can also,optionally, be encoded by the expression vector, operably linked to thecoding sequence of interest, so that the expressed polypeptide can besecreted by the recombinant host cell, for more facile isolation of thepolypeptide of interest from the cell, if desired. Such techniques arewell known in the art. (E.g., Goodey, Andrew R.; et al., Peptide and DNAsequences, U.S. Pat. No. 5,302,697; Weiner et al., Compositions andmethods for protein secretion, U.S. Pat. No. 6,022,952 and U.S. Pat. No.6,335,178; Uemura et al., Protein expression vector and utilizationthereof, U.S. Pat. No. 7,029,909; Ruben et al., 27 human secretedproteins, US 2003/0104400 A1).

The terms “in operable combination”, “in operable order” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced. Forexample, a control sequence in a vector that is “operably linked” to aprotein coding sequence is ligated thereto so that expression of theprotein coding sequence is achieved under conditions compatible with thetranscriptional activity of the control sequences.

The term “host cell” means a cell that has been transformed, or iscapable of being transformed, with a nucleic acid and thereby expressesa gene of interest. The term includes the progeny of the parent cell,whether or not the progeny is identical in morphology or in geneticmake-up to the original parent cell, so long as the gene of interest ispresent. Any of a large number of available and well-known host cellsmay be used in the practice of this invention. The selection of aparticular host is dependent upon a number of factors recognized by theart. These include, for example, compatibility with the chosenexpression vector, toxicity of the peptides encoded by the DNA molecule,rate of transformation, ease of recovery of the peptides, expressioncharacteristics, bio-safety and costs. A balance of these factors mustbe struck with the understanding that not all hosts may be equallyeffective for the expression of a particular DNA sequence. Within thesegeneral guidelines, useful microbial host cells in culture includebacteria (such as Escherichia coli sp.), yeast (such as Saccharomycessp.) and other fungal cells, insect cells, plant cells, mammalian(including human) cells, e.g., CHO cells and HEK-293 cells.Modifications can be made at the DNA level, as well. Thepeptide-encoding DNA sequence may be changed to codons more compatiblewith the chosen host cell. For E. coli, optimized codons are known inthe art. Codons can be substituted to eliminate restriction sites or toinclude silent restriction sites, which may aid in processing of the DNAin the selected host cell. Next, the transformed host is cultured andpurified. Host cells may be cultured under conventional fermentationconditions so that the desired compounds are expressed. Suchfermentation conditions are well known in the art.

The term “transfection” means the uptake of foreign or exogenous DNA bya cell, and a cell has been “transfected” when the exogenous DNA hasbeen introduced inside the cell membrane. A number of transfectiontechniques are well known in the art and are disclosed herein. See,e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001,Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, BasicMethods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.Such techniques can be used to introduce one or more exogenous DNAmoieties into suitable host cells.

The term “transformation” refers to a change in a cell's geneticcharacteristics, and a cell has been transformed when it has beenmodified to contain new DNA or RNA. For example, a cell is transformedwhere it is genetically modified from its native state by introducingnew genetic material via transfection, transduction, or othertechniques. Following transfection or transduction, the transforming DNAmay recombine with that of the cell by physically integrating into achromosome of the cell, or may be maintained transiently as an episomalelement without being replicated, or may replicate independently as aplasmid. A cell is considered to have been “stably transformed” when thetransforming DNA is replicated with the division of the cell.

By “physiologically acceptable salt” of a composition of matter, forexample a salt of the immunoglobulin, such as an antibody, is meant anysalt or salts that are known or later discovered to be pharmaceuticallyacceptable. Some non-limiting examples of pharmaceutically acceptablesalts are: acetate; trifluoroacetate; hydrohalides, such ashydrochloride and hydrobromide; sulfate; citrate; maleate; tartrate;glycolate; gluconate; succinate; mesylate; besylate; salts of gallicacid esters (gallic acid is also known as 3,4,5 trihydroxybenzoic acid)such as PentaGalloylGlucose (PGG) and epigallocatechin gallate (EGCG),salts of cholesteryl sulfate, pamoate, tannate and oxalate salts.

A “domain” or “region” (used interchangeably herein) of a protein is anyportion of the entire protein, up to and including the complete protein,but typically comprising less than the complete protein. A domain can,but need not, fold independently of the rest of the protein chain and/orbe correlated with a particular biological, biochemical, or structuralfunction or location (e.g., a ligand binding domain, or a cytosolic,transmembrane or extracellular domain).

“Treatment” or “treating” is an intervention performed with theintention of preventing the development or altering the pathology of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. “Treatment” includes any indicia of successin the amelioration of an injury, pathology or condition, including anyobjective or subjective parameter such as abatement; remission;diminishing of symptoms or making the injury, pathology or conditionmore tolerable to the patient; slowing in the rate of degeneration ordecline; making the final point of degeneration less debilitating;improving a patient's physical or mental well-being. The treatment oramelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination,self-reporting by a patient, neuropsychiatric exams, and/or apsychiatric evaluation.

An “effective amount” is generally an amount sufficient to reduce theseverity and/or frequency of symptoms, eliminate the symptoms and/orunderlying cause, prevent the occurrence of symptoms and/or theirunderlying cause, and/or improve or remediate the damage that resultsfrom or is associated with migraine headache. In some embodiments, theeffective amount is a therapeutically effective amount or aprophylactically effective amount. A “therapeutically effective amount”is an amount sufficient to remedy a disease state (e.g., transplantrejection or GVHD, inflammation, multiple sclerosis, cancer, diabetes,neuropathy, pain) or symptom(s), particularly a state or symptom(s)associated with the disease state, or otherwise prevent, hinder, retardor reverse the progression of the disease state or any other undesirablesymptom associated with the disease in any way whatsoever (i.e. thatprovides “therapeutic efficacy”). A “prophylactically effective amount”is an amount of a pharmaceutical composition that, when administered toa subject, will have the intended prophylactic effect, e.g., preventingor delaying the onset (or reoccurrence) of migraine headache or multiplesclerosis symptoms, or reducing the likelihood of the onset (orreoccurrence) of migraine headache, migraine headache symptoms, ormultiple sclerosis symptoms. The full therapeutic or prophylactic effectdoes not necessarily occur by administration of one dose, and may occuronly after administration of a series of doses. Thus, a therapeuticallyor prophylactically effective amount may be administered in one or moreadministrations.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, rats, mice, monkeys, etc.Preferably, the mammal is human.

The term “naturally occurring” as used throughout the specification inconnection with biological materials such as polypeptides, nucleicacids, host cells, and the like, refers to materials which are found innature.

The term “antibody”, or interchangeably “Ab”, is used in the broadestsense and includes fully assembled antibodies, monoclonal antibodies(including human, humanized or chimeric antibodies), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments that can bind antigen (e.g., Fab, Fab′, F(ab′)₂, Fv,single chain antibodies, diabodies), comprising complementaritydetermining regions (CDRs) of the foregoing as long as they exhibit thedesired biological activity. Multimers or aggregates of intact moleculesand/or fragments, including chemically derivatized antibodies, arecontemplated. Antibodies of any isotype class or subclass, includingIgG, IgM, IgD, IgA, and IgE, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, orany allotype, are contemplated. Different isotypes have differenteffector functions; for example, IgG1 and IgG3 isotypes haveantibody-dependent cellular cytotoxicity (ADCC) activity.

The term “antigen binding protein” (ABP) includes antibodies or antibodyfragments, as defined above, and recombinant peptides or other compoundsthat contain sequences derived from CDRs having the desiredantigen-binding properties such that they specifically bind a targetantigen of interest.

In general, an antigen binding protein, e.g., an antibody or antibodyfragment, “specifically binds” to an antigen of interest (e.g., IL-17Ror TR2) when it has a significantly higher binding affinity for, andconsequently is capable of distinguishing, that antigen, compared to itsaffinity for other unrelated proteins, under similar binding assayconditions. Typically, an antigen binding protein is said to“specifically bind” its target antigen when the dissociation constant(K_(D)) is ≦10⁻⁸ M. The antibody specifically binds antigen with “highaffinity” when the K_(D) is ≦5×10⁻⁹ M, and with “very high affinity”when the K_(D) is ≦5×10⁻¹⁰ M. In one embodiment, the antibodies willbind to the antigen of interest with a K_(D) of between about 10⁻⁸ M and10⁻¹⁰ M, and in yet another embodiment the antibodies will bind with aK_(D)≦5×10⁻⁹.

“Antigen binding region” or “antigen binding site” means a portion of aprotein, that specifically binds a specified antigen, e.g., IL-17R orTR2. For example, that portion of an antigen binding protein thatcontains the amino acid residues that interact with an antigen andconfer on the antigen binding protein its specificity and affinity forthe antigen is referred to as “antigen binding region.” An antigenbinding region typically includes one or more “complementary bindingregions” (“CDRs”). Certain antigen binding regions also include one ormore “framework” regions (“FRs”). A “CDR” is an amino acid sequence thatcontributes to antigen binding specificity and affinity. “Framework”regions can aid in maintaining the proper conformation of the CDRs topromote binding between the antigen binding region and an antigen. In atraditional antibody, the CDRs are embedded within a framework in theheavy and light chain variable region where they constitute the regionsresponsible for antigen binding and recognition. A variable region of animmunoglobulin antigen binding protein comprises at least three heavy orlight chain CDRs, see, supra (Kabat et al., 1991, Sequences of Proteinsof Immunological Interest, Public Health Service N.I.H., Bethesda, Md.;see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia etal., 1989, Nature 342: 877-883), within a framework region (designatedframework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991,supra; see also Chothia and Lesk, 1987, supra).

An “isolated” immunoglobulin, e.g., an antibody or antibody fragment, isone that has been identified and separated from one or more componentsof its natural environment or of a culture medium in which it has beensecreted by a producing cell. “Contaminant” components of its naturalenvironment or medium are materials that would interfere with diagnosticor therapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. In someembodiments, the antibody will be purified (1) to greater than 95% byweight of antibody, and most preferably more than 99% by weight, or (2)to homogeneity by SDS-PAGE under reducing or nonreducing conditions,optionally using a stain, e.g., Coomassie blue or silver stain. Isolatednaturally occurring antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Typically, however, isolated antibodywill be prepared by at least one purification step.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies that are antigen binding proteinsare highly specific binders, being directed against an individualantigenic site or epitope, in contrast to polyclonal antibodypreparations that typically include different antibodies directedagainst different epitopes. Nonlimiting examples of monoclonalantibodies include murine, rabbit, rat, chicken, chimeric, humanized, orhuman antibodies, fully assembled antibodies, multispecific antibodies(including bispecific antibodies), antibody fragments that can bind anantigen (including, Fab, Fab′, F(ab′)₂, Fv, single chain antibodies,diabodies), maxibodies, nanobodies, and recombinant peptides comprisingCDRs of the foregoing as long as they exhibit the desired biologicalactivity, or variants or derivatives thereof.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature,256:495 [1975], or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature, 352:624-628[1991] and Marks et al., J. Mol.Biol., 222:581-597 (1991), for example.

A “multispecific” binding agent or antigen binding protein or antibodyis one that targets more than one antigen or epitope.

A “bispecific,” “dual-specific” or “bifunctional” binding agent orantigen binding protein or antibody is a hybrid having two differentantigen binding sites. Biantigen binding proteins, antigen bindingproteins and antibodies are a species of multiantigen binding protein,antigen binding protein or multispecific antibody and may be produced bya variety of methods including, but not limited to, fusion of hybridomasor linking of Fab′ fragments. See, e.g., Songsivilai and Lachmann, 1990,Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol.148:1547-1553. The two binding sites of a bispecific antigen bindingprotein or antibody will bind to two different epitopes, which mayreside on the same or different protein targets.

The term “immunoglobulin” encompasses full antibodies comprising twodimerized heavy chains (HC), each covalently linked to a light chain(LC); a single undimerized immunoglobulin heavy chain and covalentlylinked light chain (HC+LC), or a chimeric immunoglobulin (lightchain+heavy chain)-Fc heterotrimer (a so-called “hemibody”). An“immunoglobulin” is a protein, but is not necessarily an antigen bindingprotein.

In an “antibody”, each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” chain of about 220amino acids (about 25 kDa) and one “heavy” chain of about 440 aminoacids (about 50-70 kDa). The amino-terminal portion of each chainincludes a “variable” (“V”) region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. The variable region differsamong different antibodies. The constant region is the same amongdifferent antibodies. Within the variable region of each heavy or lightchain, there are three hypervariable subregions that help determine theantibody's specificity for antigen in the case of an antibody that is anantigen binding protein. However, within the scope of the presentinvention, an embodiment of the immunoglobulin, e.g., an antibody, neednot be an antigen binding protein, or need not be known to specificallybind to an antigen. The variable domain residues between thehypervariable regions are called the framework residues and generallyare somewhat homologous among different antibodies. Immunoglobulins canbe assigned to different classes depending on the amino acid sequence ofthe constant domain of their heavy chains. Human light chains areclassified as kappa (κ) and lambda (λ) light chains. Within light andheavy chains, the variable and constant regions are joined by a “J”region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)). Within the scope of the invention, an “antibody” alsoencompasses a recombinantly made antibody, and antibodies that areglycosylated or lacking glycosylation.

The term “light chain” or “immunoglobulin light chain” includes afull-length light chain and fragments thereof having sufficient variableregion sequence to confer binding specificity. A full-length light chainincludes a variable region domain, V_(L), and a constant region domain,C_(L). The variable region domain of the light chain is at theamino-terminus of the polypeptide. Light chains include kappa chains andlambda chains.

The term “heavy chain” or “immunoglobulin heavy chain” includes afull-length heavy chain and fragments thereof having sufficient variableregion sequence to confer binding specificity. A full-length heavy chainincludes a variable region domain, V_(H), and three constant regiondomains, C_(H)1, C_(H)2, and C_(H)3. The V_(H) domain is at theamino-terminus of the polypeptide, and the C_(H) domains are at thecarboxyl-terminus, with the C_(H)3 being closest to the carboxy-terminusof the polypeptide. Heavy chains are classified as mu (μ), delta (Δ),gamma (γ), alpha (α), and epsilon (ε), and define the antibody's isotypeas IgM, IgD, IgG, IgA, and IgE, respectively. In separate embodiments ofthe invention, heavy chains may be of any isotype, including IgG(including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 andIgA2 subtypes), IgM and IgE. Several of these may be further dividedinto subclasses or isotypes, e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.Different IgG isotypes may have different effector functions (mediatedby the Fc region), such as antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fcregion of an antibody binds to Fc receptors (FcγRs) on the surface ofimmune effector cells such as natural killers and macrophages, leadingto the phagocytosis or lysis of the targeted cells. In CDC, theantibodies kill the targeted cells by triggering the complement cascadeat the cell surface.

An “Fc region”, or used interchangeably herein, “Fc domain” or“immunoglobulin Fc domain”, contains two heavy chain fragments, which ina full antibody comprise the C_(H)1 and C_(H)2 domains of the antibody.The two heavy chain fragments are held together by two or more disulfidebonds and by hydrophobic interactions of the C_(H)3 domains.

The term “salvage receptor binding epitope” refers to an epitope of theFc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that isresponsible for increasing the in vivo serum half-life of the IgGmolecule.

“Allotypes” are variations in antibody sequence, often in the constantregion, that can be immunogenic and are encoded by specific alleles inhumans. Allotypes have been identified for five of the human IGHC genes,the IGHG1, IGHG2, IGHG3, IGHA2 and IGHE genes, and are designated asG1m, G2m, G3m, A2m, and Em allotypes, respectively. At least 18 Gmallotypes are known: nG1m(1), nG1m(2), G1m (1, 2, 3, 17) or G1m (a, x,f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24,26, 27, 28) or G3m (b1, c3, b5, b0, b3, b4, s, t, g1, c5, u, v, g5).There are two A2m allotypes A2m(1) and A2m(2).

For a detailed description of the structure and generation ofantibodies, see Roth, D. B., and Craig, N. L., Cell, 94:411-414 (1998),herein incorporated by reference in its entirety. Briefly, the processfor generating DNA encoding the heavy and light chain immunoglobulinsequences occurs primarily in developing B-cells. Prior to therearranging and joining of various immunoglobulin gene segments, the V,D, J and constant (C) gene segments are found generally in relativelyclose proximity on a single chromosome. During B-cell-differentiation,one of each of the appropriate family members of the V, D, J (or only Vand J in the case of light chain genes) gene segments are recombined toform functionally rearranged variable regions of the heavy and lightimmunoglobulin genes. This gene segment rearrangement process appears tobe sequential. First, heavy chain D-to-J joints are made, followed byheavy chain V-to-DJ joints and light chain V-to-J joints. In addition tothe rearrangement of V, D and J segments, further diversity is generatedin the primary repertoire of immunoglobulin heavy and light chains byway of variable recombination at the locations where the V and Jsegments in the light chain are joined and where the D and J segments ofthe heavy chain are joined. Such variation in the light chain typicallyoccurs within the last codon of the V gene segment and the first codonof the J segment. Similar imprecision in joining occurs on the heavychain chromosome between the D and J_(H) segments and may extend over asmany as 10 nucleotides. Furthermore, several nucleotides may be insertedbetween the D and J_(H) and between the V_(H) and D gene segments whichare not encoded by genomic DNA. The addition of these nucleotides isknown as N-region diversity. The net effect of such rearrangements inthe variable region gene segments and the variable recombination whichmay occur during such joining is the production of a primary antibodyrepertoire.

The term “hypervariable” region refers to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from a complementarity determiningregion or CDR [i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) inthe light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain as described by Kabat et al.,Sequences of Proteins of Immunological Interest, 5^(th) Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)].Even a single CDR may recognize and bind antigen, although with a loweraffinity than the entire antigen binding site containing all of theCDRs.

An alternative definition of residues from a hypervariable “loop” isdescribed by Chothia et al., J. Mol. Biol. 196: 901-917 (1987) asresidues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain.

“Framework” or “FR” residues are those variable region residues otherthan the hypervariable region residues.

“Antibody fragments” comprise a portion of an intact full lengthantibody, preferably the antigen binding or variable region of theintact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)₂, and Fv fragments; diabodies; linear antibodies (Zapata et al.,Protein Eng.,8(10):1057-1062 (1995)); single-chain antibody molecules;and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment which contains the constant region.The Fab fragment contains all of the variable domain, as well as theconstant domain of the light chain and the first constant domain (CH1)of the heavy chain. The Fc fragment displays carbohydrates and isresponsible for many antibody effector functions (such as bindingcomplement and cell receptors), that distinguish one class of antibodyfrom another.

Pepsin treatment yields an F(ab′)₂ fragment that has two “Single-chainFv” or “scFv” antibody fragments comprising the VH and VL domains ofantibody, wherein these domains are present in a single polypeptidechain. Fab fragments differ from Fab′ fragments by the inclusion of afew additional residues at the carboxy terminus of the heavy chain CH1domain including one or more cysteines from the antibody hinge region.Preferably, the Fv polypeptide further comprises a polypeptide linkerbetween the VH and VL domains that enables the Fv to form the desiredstructure for antigen binding. For a review of scFv see Pluckthun in ThePharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994).

A “Fab fragment” is comprised of one light chain and the C_(H)1 andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the V_(H) domain and the C_(H)1 domain and also theregion between the C_(H)1 and C_(H)2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form an F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)2 domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the VH VL dimer. A single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen, although at a lower affinity than the entirebinding site.

“Single-chain antibodies” are Fv molecules in which the heavy and lightchain variable regions have been connected by a flexible linker to forma single polypeptide chain, which forms an antigen-binding region.Single chain antibodies are discussed in detail in International PatentApplication Publication No. WO 88/01649 and U.S. Pat. No. 4,946,778 andNo. 5,260,203, the disclosures of which are incorporated by reference intheir entireties.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain, and optionally comprising a polypeptide linkerbetween the V_(H) and V_(L) domains that enables the Fv to form thedesired structure for antigen binding (Bird et al., Science 242:423-426,1988, and Huston et al., Proc. Nati. Acad. Sci. USA 85:5879-5883, 1988).An “Fd” fragment consists of the V_(H) and C_(H)1 domains.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody maytarget the same or different antigens.

The term “compete” when used in the context of antigen binding proteins(e.g., neutralizing antigen binding proteins or neutralizing antibodies)that compete for the same epitope means competition between antigenbinding proteins is determined by an assay in which the antigen bindingprotein (e.g., antibody or immunologically functional fragment thereof)under test prevents or inhibits specific binding of a reference antigenbinding protein (e.g., a ligand, or a reference antibody) to a commonantigen (e.g., IL-17R or a fragment thereof, or TR2 or a fragmentthereof). Numerous types of competitive binding assays can be used, forexample: solid phase direct or indirect radioimmunoassay (RIA), solidphase direct or indirect enzyme immunoassay (EIA), sandwich competitionassay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253);solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986,J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phasedirect labeled sandwich assay (see, e.g., Harlow and Lane, 1988,Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phasedirect label RIA using I-125 label (see, e.g., Morel et al., 1988,Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see,e.g., Cheung, et al., 1990, Virology 176:546-552); direct labeled RIA(Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82); and surfaceplasmon resonance (BIAcore®; e.g., Fischer et al., Apeptide-immunoglobulin-conjugate, WO 2007/045463 A1, Example 10, whichis incorporated herein by reference in its entirety), or KinExA.Typically, such an assay involves the use of purified antigen bound to asolid surface or cells bearing either of these, an unlabelled testimmunoglobulin or antigen binding protein and a labeled referenceantigen binding protein. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test antigen binding protein. Usually the testimmunoglobulin or antigen binding protein is present in excess. Antigenbinding proteins identified by competition assay (competing antigenbinding proteins) include antigen binding proteins binding to the sameepitope as the reference antigen binding proteins and antigen bindingproteins binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antigen binding protein for sterichindrance to occur. Additional details regarding methods for determiningcompetitive binding are provided in the examples herein. Usually, when acompeting antigen binding protein is present in excess, it will inhibitspecific binding of a reference antigen binding protein to a commonantigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In someinstance, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% ormore.

When an immunoglobulin (e.g., an antibody or antibody fragment) “doesnot significantly bind” an antigen it means that the particularimmunoglobulin, in excess, does not compete with a reference antigenbinding protein, e.g., with a positive control antibody, to inhibit itsbinding to the target antigen by >39%, or >30%, or >20%, or >10%. As tospecific binding to soluble human IL-17R, a positive control antibody isantibody 16429, described herein. As to specific binding to solublehuman TR2, a positive control antibody is antibody 16449, describedherein.

Antibody-antigen interactions can be characterized by the associationrate constant in M⁻¹s⁻¹ (k_(a)), or the dissociation rate constant ins⁻¹ (k_(d)), or alternatively the dissociation equilibrium constant in M(K_(D)). Association rate constants, dissociation rate constants, ordissociation equilibrium constants may be readily determined usingkinetic analysis techniques such as surface plasmon resonance (BIAcore®;e.g., Fischer et al., A peptide-immunoglobulin-conjugate, WO 2007/045463A1, Example 10, which is incorporated herein by reference in itsentirety), or KinExA using general procedures outlined by themanufacturer or other methods known in the art. The kinetic dataobtained by BIAcore® or KinExA may be analyzed by methods described bythe manufacturer.

“Measured by a surface plasmon resonance binding assay” with respect todetermining whether a test immunoglobulin “does not significantly bind”means as measured in the solution equilibrium binding assay describedherein to assess the binding activity of immunoglobulins based onsurface plasmon resonance. A reference antigen binding protein (e.g.,Antibody 16429 for human IL-17R or Antibody 16449 for human TR2) isimmobilized to a BIACore® 2000, research grade sensor chip CM5 surfaceaccording to manufacturer's instructions (BIACore, Inc., Piscataway,N.J.). Carboxyl groups on the sensor chip surfaces are activated byinjecting 60 μL of a mixture containing 0.2 MN-ethyl-N′-(dimethylaminopropyl) carbodiimide (EDC) and 0.05 MN-hydroxysuccinimide (NHS). The reference antigen binding protein isdiluted in 10 mM sodium acetate, pH 4.0 and injected over the activatedchip surface at 30 μL/min for 6 minutes. Excess reactive groups on thesurfaces are deactivated by injecting 60 μL of 1 M ethanolamine. Thefinal immobilized level is typically approximately 6600 resonance units(RU). Soluble target antigen (e.g., 10 nM of soluble human IL-17R or 30nM of soluble human TR2) in the absence of soluble antigen bindingprotein (e.g., antibody) is used to establish the 100% binding signal tothe fixed reference antigen binding protein (e.g., the positive controlantibody). The decreased binding signal of the target antigen afterincubation of the test immunoglobulin indicates its level of binding tothe target antigen in solution.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as an antigenbinding protein (including, e.g., an antibody or immunologicalfunctional fragment thereof), and additionally capable of being used inan animal to produce antibodies capable of binding to that antigen. Anantigen may possess one or more epitopes that are capable of interactingwith different antigen binding proteins, e.g., antibodies.

The term “epitope” is the portion of a molecule that is bound by anantigen binding protein (for example, an antibody). The term includesany determinant capable of specifically binding to an antigen bindingprotein, such as an antibody or to a T-cell receptor. An epitope can becontiguous or non-contiguous (e.g., in a single-chain polypeptide, aminoacid residues that are not contiguous to one another in the polypeptidesequence but that within the context of the molecule are bound by theantigen binding protein). In certain embodiments, epitopes may bemimetic in that they comprise a three dimensional structure that issimilar to an epitope used to generate the antigen binding protein, yetcomprise none or only some of the amino acid residues found in thatepitope used to generate the antigen binding protein. Most often,epitopes reside on proteins, but in some instances may reside on otherkinds of molecules, such as nucleic acids. Epitope determinants mayinclude chemically active surface groupings of molecules such as aminoacids, sugar side chains, phosphoryl or sulfonyl groups, and may havespecific three dimensional structural characteristics, and/or specificcharge characteristics. Generally, antibodies specific for a particulartarget antigen will preferentially recognize an epitope on the targetantigen in a complex mixture of proteins and/or macromolecules.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by aligning and comparing the sequences. “Percentidentity” means the percent of identical residues between the aminoacids or nucleotides in the compared molecules and is calculated basedon the size of the smallest of the molecules being compared. For thesecalculations, gaps in alignments (if any) must be addressed by aparticular mathematical model or computer program (i.e., an“algorithm”). Methods that can be used to calculate the identity of thealigned nucleic acids or polypeptides include those described inComputational Molecular Biology, (Lesk, A. M., ed.), 1988, New York:Oxford University Press; Biocomputing Informatics and Genome Projects,(Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysisof Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.),1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysisin Molecular Biology, New York: Academic Press; Sequence AnalysisPrimer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.For example, sequence identity can be determined by standard methodsthat are commonly used to compare the similarity in position of theamino acids of two polypeptides. Using a computer program such as BLASTor FASTA, two polypeptide or two polynucleotide sequences are alignedfor optimal matching of their respective residues (either along the fulllength of one or both sequences, or along a pre-determined portion ofone or both sequences). The programs provide a default opening penaltyand a default gap penalty, and a scoring matrix such as PAM 250 [astandard scoring matrix; see Dayhoff et al., in Atlas of ProteinSequence and Structure, vol. 5, supp. 3 (1978)] can be used inconjunction with the computer program. For example, the percent identitycan then be calculated as: the total number of identical matchesmultiplied by 100 and then divided by the sum of the length of thelonger sequence within the matched span and the number of gapsintroduced into the longer sequences in order to align the twosequences. In calculating percent identity, the sequences being comparedare aligned in a way that gives the largest match between the sequences.

The GCG program package is a computer program that can be used todetermine percent identity, which package includes GAP (Devereux et al.,1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University ofWisconsin, Madison, Wis.). The computer algorithm GAP is used to alignthe two polypeptides or two polynucleotides for which the percentsequence identity is to be determined. The sequences are aligned foroptimal matching of their respective amino acid or nucleotide (the“matched span”, as determined by the algorithm). A gap opening penalty(which is calculated as 3× the average diagonal, wherein the “averagediagonal” is the average of the diagonal of the comparison matrix beingused; the “diagonal” is the score or number assigned to each perfectamino acid match by the particular comparison matrix) and a gapextension penalty (which is usually 1/10 times the gap opening penalty),as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used inconjunction with the algorithm. In certain embodiments, a standardcomparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequenceand Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff etal., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM62 comparison matrix) is also used by the algorithm.

Recommended parameters for determining percent identity for polypeptidesor nucleotide sequences using the GAP program include the following:

Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;

Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;

Gap Penalty: 12 (but with no penalty for end gaps)

Gap Length Penalty: 4

Threshold of Similarity: 0

Certain alignment schemes for aligning two amino acid sequences mayresult in matching of only a short region of the two sequences, and thissmall aligned region may have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (GAP program) canbe adjusted if so desired to result in an alignment that spans at least50 contiguous amino acids of the target polypeptide.

The term “modification” when used in connection with immunoglobulins,including antibodies and antibody fragments, of the invention, include,but are not limited to, one or more amino acid changes (includingsubstitutions, insertions or deletions); chemical modifications;covalent modification by conjugation to therapeutic or diagnosticagents; labeling (e.g., with radionuclides or various enzymes); covalentpolymer attachment such as PEGylation (derivatization with polyethyleneglycol) and insertion or substitution by chemical synthesis ofnon-natural amino acids. Modified immunoglobulins of the invention willretain the binding (or non-binding) properties of unmodified moleculesof the invention.

The term “derivative” when used in connection with immunoglobulins(including antibodies and antibody fragments) of the invention refers toimmunoglobulins that are covalently modified by conjugation totherapeutic or diagnostic agents, labeling (e.g., with radionuclides orvarious enzymes), covalent polymer attachment such as PEGylation(derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of non-natural amino acids. Derivatives of theinvention will retain the binding properties of underivatized moleculesof the invention.

Embodiments of Immunoglobulins

In full-length immunoglobulin light and heavy chains, the variable andconstant regions are joined by a “J” region of about twelve or moreamino acids, with the heavy chain also including a “D” region of aboutten more amino acids. See, e.g., Fundamental Immunology, 2nd ed., Ch. 7(Paul, W., ed.) 1989, New York: Raven Press (hereby incorporated byreference in its entirety for all purposes). The variable regions ofeach light/heavy chain pair typically form the antigen binding site.

One example of a human IgG2 heavy chain (HC) constant domain has theamino acid sequence:

SEQ. ID NO: 86 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//.

Constant region sequences of other IgG isotypes are known in the art formaking recombinant versions of the inventive immunoglobulin having anIgG1, IgG2, IgG3, or IgG4 immunoglobulin isotype, if desired. Ingeneral, human IgG2 can be used for targets where effector functions arenot desired, and human IgG1 in situations where such effector functions(e.g., antibody-dependent cytotoxicity (ADCC)) are desired. Human IgG3has a relatively short half life and human IgG4 forms antibody“half-molecules.” There are four known allotypes of human IgG1. Thepreferred allotype is referred to as “hIgG1z”, also known as the “KEEM”allotype. Human IgG1 allotypes “hIgG1za” (KDEL), “hIgG1f” (REEM), and“hIgG1fa” are also useful; all appear to have ADCC effector function.

Human hIgG1z heavy chain (HC) constant domain has the amino acidsequence:

SEQ ID NO: 87 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//.

Human hIgG1za heavy chain (HC) constant domain has the amino acidsequence:

SEQ ID NO: 88 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//.

Human hIgG1f heavy chain (HC) constant domain has the amino acidsequence:

SEQ ID NO: 127 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//.

Human hIgG1fa heavy chain (HC) constant domain has the amino acidsequence:

SEQ ID NO: 90 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//.

One example of a human immunoglobulin light chain (LC) constant regionsequence is the following (designated “CL-1”):

SEQ ID NO: 91 GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK TVAPTECS//.

CL-1 is useful to increase the pI of antibodies and is convenient. Thereare three other human immunoglobulin light chain constant regions,designated “CL-2”, “CL-3” and “CL-7”, which can also be used within thescope of the present invention. CL-2 and CL-3 are more common in thehuman population. CL-2 human light chain (LC) constant domain has theamino acid sequence:

SEQ ID NO: 92 Gqpkaapsvtlfppsseelqankatlvclisdfypgavtvawkadsspvkagvetttpskqsnnkyaassylsltpeqwkshrsyscqvthegstvektv aptecs//.

CL-3 human LC constant domain has the amino acid sequence:

SEQ ID NO: 93 gqpkaapsvtlfppsseelqankatlvclisdfypgavtvawkadsspvkagvetttpskqsnnkyaassylsltpeqwkshksyscqvthegstvektv aptecs//.

CL-7 human LC constant domain has the amino acid sequence:

SEQ ID NO: 94 Gqpkaapsvtlfppsseelqankatlvclvsdfypgavtvawkadgspvkvgvettkpskqsnnkyaassylsltpeqwkshrsyscrvthegstvektv apaecs//.

Human LC kappa constant region has the amino acid sequence:

SEQ ID NO: 129 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC//.

Variable regions of immunoglobulin chains generally exhibit the sameoverall structure, comprising relatively conserved framework regions(FR) joined by three hypervariable regions, more often called“complementarity determining regions” or CDRs. The CDRs from the twochains of each heavy chain/light chain pair mentioned above typicallyare aligned by the framework regions to form a structure that bindsspecifically with a specific epitope or domain on the target (e.g.,human IL-17R or human TR2), however within the scope of the presentinvention, the original CDR sequences have been deliberately modified soas not significantly to bind to human IL-17R or TR2 targets. FromN-terminal to C-terminal, naturally-occurring light and heavy chainvariable regions both typically conform with the following order ofthese elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numberingsystem has been devised for assigning numbers to amino acids that occupypositions in each of these domains. This numbering system is defined inKabat Sequences of Proteins of Immunological Interest (1987 and 1991,NIH, Bethesda, Md.), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917;Chothia et al., 1989, Nature 342:878-883.

Specific examples of some of the full length light and heavy chains ofthe antibodies that are provided and their corresponding amino acidsequences are summarized in Table 1A and Table 1B below. Table 1A showsexemplary light chain sequences. Table 1B shows exemplary heavy chainsequences, some of which include constant region human IgG2 (SEQ IDNO:86) and some of which include constant region human IgG1f (SEQ IDNO:127). However, encompassed within the present invention areimmunoglobulins with sequence changes in the constant or frameworkregions of those listed in Table 1A and/or Table 1B (e.g. IgG4 vs IgG2,CL2 vs CL1). Also, signal peptide (SP) sequences for all of the sequencein Table 1A and Table 1B are included, such as, the VK-1 SP signalpeptide: MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO:103), MEAPAQLLFLLLLWLPDTTG(SEQ ID NO:104), MEWTWRVLFLVAAATGAHS (SEQ ID NO:105),METPAQLLFLLLLWLPDTTG (SEQ ID NO:106), MKHLWFFLLLVAAPRWVLS (SEQ IDNO:107), but any other suitable signal peptide sequence may be employedwithin the scope of the invention. Another example of a useful signalpeptide sequence is VH21 SP MEWS WVFLFFLSVTTGVHS (SEQ ID NO:95). Otherexemplary signal peptide sequences are shown in Table 1A-B.

TABLE 1A Immunoglobulin Light Chain Sequences. Signalpeptide sequences are indicated by underline. SEQ ID NO: DesignationSequence 109 16435 MEAPAQLLFLLLLWLPDTTGEIVMTQSPATLSV (LC: P66L,SPGERATLSCRASQSVSSNLAWFQQKPGQAPR D90E)LLIYDASTRATGVPARFSGSGSGTEFTLTISSLQ SEDFAVYYCQQYDNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 110 16435EIVMTQSPATLSVSPGERATLSCRASQSVSSNL (LC: P66L,AWFQQKPGQAPRLLIYDASTRATGVPARFSGS D90E) GSGTEFTLTISSLQSEDFAVYYCQQYDNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 121 16444MEAPAQLLFLLLLWLPDTTGEIVMTQSPATLSV (LC: P66L,SPGERATLSCRASQSVSSNLAWFQQKPGQAPR D90E,LLIYDASTRATGVPARFSGSGSGTEFTLTISSLQ W114A)SEDFAVYYCQQYDNAPLTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC122 16444 EIVMTQSPATLSVSPGERATLSCRASQSVSSNL (LC: P66L,AWFQQKPGQAPRLLIYDASTRATGVPARFSGS D90E, GSGTEFTLTISSLQSEDFAVYYCQQYDNAPLTFW114A) GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC  97 4241 METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLS(LC: Y53A) PGERATLSCRASQGISRSALAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPE DFAVYYCQQFGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC  98 4241EIVLTQSPGTLSLSPGERATLSCRASQGISRSAL (LC: Y53A)AWYQQKPGQAPSLLIYGASSRATGIPDRFSGSG SGTDFTLTISRLEPEDFAVYYCQQFGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 115 4341METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLS (LC: Y53E)PGERATLSCRASQGISRSELAWYQQKPGQAPSL LIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFGSSPWTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC116 4341 EIVLTQSPGTLSLSPGERATLSCRASQGISRSELA (LC: Y53E)WYQQKPGQAPSLLIYGASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYYCQQFGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC

TABLE 1B Immunoglobulin Heavy Chain Sequences. Signalpeptide sequences are indicated by underline. SEQ ID NO: DesignationSequence 112 16435 MEWTWRVLFLVAAATGAHSQVQLVQSGA (HC: R118A)EVKKPGASVKVSCKASGYTFTRYGISWVRQ APGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR AQLYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 113 16435 QVQLVQSGAEVKKPGASVKVSCKASGYTF (HC: R118A)TRYGISWVRQAPGQGLEWMGWISTYSGNT NYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARAQLYFDYWGQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVV SVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 124 16444MEWTWRVLFLVAAATGAHSQVQLVQSGA (HC: R118A, EVKKPGASVKVSCKASGYTFTRYGISWVRQL120Q) APGQGLEWMGWISTYSGNTNYAQKLQGRV TMTTDTSTSTAYMELRSLRSDDTAVYYCARAQQYFDYWGQGTLVTVSSASTKGPSVFPLA PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWL NGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 125 16444QVQLVQSGAEVKKPGASVKVSCKASGYTF (HC: R118A, TRYGISWVRQAPGQGLEWMGWISTYSGNTL120Q) NYAQKLQGRVTMTTDTSTSTAYMELRSLRS DDTAVYYCARAQQYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT KVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 100 4241 MKHLWFFLLLVAAPRWVLSQVQLQESGPG(HC: Y125E) LVKPSQTLSLTCTVSGGSISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTIS VDTSKKQFSLRLSSVTAADTAVYYCARDRGGDYEYGMDVWGQGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 101 4241QVQLQESGPGLVKPSQTLSLTCTVSGGSISS (HC: Y125E)GDYFWSWIRQLPGKGLEWIGHIHNSGTTYY NPSLKSRVTISVDTSKKQFSLRLSSVTAADTAVYYCARDRGGDYEYGMDVWGQGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK 118 4341MKHLWFFLLLVAAPRWVLSQVQLQESGPG (HC: Y125A)LVKPSQTLSLTCTVSGGSISSGDYFWSWIRQ LPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLRLSSVTAADTAVYYCARDRG GDYAYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 119 4341 QVQLQESGPGLVKPSQTLSLTCTVSGGSISS(HC: Y125A) GDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLRLSSVTAADT AVYYCARDRGGDYAYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Some useful embodiments of the isolated immunoglobulin comprising anantibody or antibody fragment, comprise:

(a) an immunoglobulin heavy chain comprising the amino acid sequence ofSEQ ID NO:113, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminalor C-terminal, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:110, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminal or C-terminal, or both; or

(b) an immunoglobulin heavy chain comprising the amino acid sequence ofSEQ ID NO:125, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminalor C-terminal, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:122, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminal or C-terminal, or both; or

(c) an immunoglobulin heavy chain comprising the amino acid sequence ofSEQ ID NO:101, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminalor C-terminal, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:98, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminal or C-terminal, or both; or

(d) an immunoglobulin heavy chain comprising the amino acid sequence ofSEQ ID NO:119, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminalor C-terminal, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:116, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminal or C-terminal, or both.

In some instances, such antibodies include at least one heavy chain andone light chain, whereas in other instances the variant forms containtwo identical light chains and two identical heavy chains. It is withinthe scope of the invention that the heavy chain(s) and/or light chain(s)may have one, two, three, four or five amino acid residues lacking fromthe N-terminal or C-terminal, or both, in relation to any one of theheavy and light chains set forth in Tables 1A and Table 1B, e.g., due topost-translational modifications. For example, CHO cells typicallycleave off a C-terminal lysine. As described herein, certain embodimentscomprising conjugates with one or more pharmacologically active chemicalmoieties, such as a phramacologically active polypeptide can compriseheteromultimers, such as monovalent heterodimers, heterotrimers, orheterotetramers, as illustrated schematically in FIGS. 1F-1N (see, alsoTable 2D).

Variable Domains of Immunogloblins, e.g., Antibodies

The various heavy chain and light chain variable regions provided hereinare depicted in Table 2A-B. Each of these variable regions may beattached to the above heavy and light chain constant regions to form acomplete antibody heavy and light chain, respectively. Further, each ofthe so generated heavy and light chain sequences may be combined to forma complete antibody structure. It should be understood that the heavychain and light chain variable regions provided herein can also beattached to other constant domains having different sequences than theexemplary sequences listed above.

Also provided are immunoglobulins, including antibodies or antibodyfragments, that contain or include at least one immunoglobulin lightchain variable region selected from V_(L)2, V_(L)3, V_(L)4, and V_(L)5,as shown in Table 2A below, and at least one immunoglobulin heavy chainvariable region selected from V_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6,V_(H)7, V_(H)8, V_(H)9, V_(H)10, and V_(H)11, as shown in Table 2Bbelow, and immunologically functional fragments, derivatives, muteinsand variants of these light chain and heavy chain variable regions.Examples of such embodiments are found in Table 2C and Table 2D below.

Also provided are immunoglobulins, including antibodies or antibodyfragments, that contain or include at least one immunoglobulin lightchain variable region selected from V_(L)7, V_(L)8, V_(L)9, V_(L)10,V_(L)11, V_(L)12, V_(L)13, V_(L)14, V_(L)15 and V_(L)16, as shown inTable 2A below, and at least one immunoglobulin heavy chain variableregion selected from V_(H)13, V_(H)14, V_(H)15, V_(H)16, V_(H)17,V_(H)18, V_(H)19, V_(H)20, V_(H)21, V_(H)22, V_(H)23, V_(H)24, V_(H)25,V_(H)26, V_(H)27, V_(H)28, V_(H)29, V_(H)30, V_(H)31, V_(H)32, V_(H)33,V_(H)34, V_(H)35, and V_(H)36, as shown in Table 2B below, andimmunologically functional fragments, derivatives, muteins and variantsof these light chain and heavy chain variable regions. Examples of suchembodiments are found in Table 2C and Table 2D below.

Exemplary embodiments of the inventive immunoglobulin include those, inwhich:

the heavy chain variable region comprises the amino acid sequence of SEQID NO:323 [VH10]; and the light chain variable region comprises theamino acid sequence of SEQ ID NO:188 [VL4]; or

the light chain variable region comprises the amino acid sequence of SEQID NO:196 [VL8]; and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:353 [VH25]; or

the light chain variable region comprises the amino acid sequence of SEQID NO:202 [VL11]; and the heavy chain variable region comprises theamino acid sequence of SEQ ID NO:349 [VH23]; or

the heavy chain variable region comprises the amino acid sequence of SEQID NO:325 [VH11]; and the light chain variable region comprises theamino acid sequence of SEQ ID NO:190 [VL5].

Immunoglobulins of this type can generally be designated by the formula“V_(H)x/V_(L)y,” where “x” corresponds to the number of heavy chainvariable regions included in the immunoglobulin and “y” corresponds tothe number of the light chain variable regions included in theimmunoglobulin (in general, x and y are each 1 or 2).

TABLE 2A Exemplary V_(L) Chains. Optional N-terminal signalsequences are not shown, but may be reflectedin the arbitrary “description” of the V_(L). SEQ Desig- ID nationDescription NO Amino Acid Sequence VL1 Anti-IL-17R 182EIVMTQSPATLSVSPGERATLSCRASQS Wild type VSSNLAWFQQKPGQAPRPLIYDASTRA (WT)TGVPARFSGSGSGTDFTLTISSLQSEDFA VYYCQQYDNWPLTFGGGTKVEIK VL2 W114A 184EIVMTQSPATLSVSPGERATLSCRASQS VSSNLAWFQQKPGQAPRPLIYDASTRATGVPARFSGSGSGTDFTLTISSLQSEDFA VYYCQQYDNAPLTFGGGTKVEIK VL3 Y111A 186EIVMTQSPATLSVSPGERATLSCRASQS VSSNLAWFQQKPGQAPRPLIYDASTRATGVPARFSGSGSGTDFTLTISSLQSEDFA VYYCQQADNWPLTFGGGTKVEIK VL4 P66L, D90E 188EIVMTQSPATLSVSPGERATLSCRASQS VSSNLAWFQQKPGQAPRLLIYDASTRATGVPARFSGSGSGTEFTLTISSLQSEDFA VYYCQQYDNWPLTFGGGTKVEIK VL5 P66L, D90E,190 EIVMTQSPATLSVSPGERATLSCRASQS W114A VSSNLAWFQQKPGQAPRLLIYDASTRATGVPARFSGSGSGTEFTLTISSLQSEDFA VYYCQQYDNAPLTFGGGTKVEIK VL6 Anti-huTR2 192EIVLTQSPGTLSLSPGERATLSCRASQGI Wild type SRSYLAWYQQKPGQAPSLLIYGASSRA (WT)TGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQFGSSPWTFGQGTKVEIK VL7 FI12A 194EIVLTQSPGTLSLSPGERATLSCRASQGI SRSYLAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQAGSSPWTFGQGTKVEIK VL8 Y53A 196EIVLTQSPGTLSLSPGERATLSCRASQGI SRSALAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQFGSSPWTFGQGTKVEIK VL9 W117A 198EIVLTQSPGTLSLSPGERATLSCRASQGI SRSYLAWYQQKPGQAPSLL1YGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQFGSSPATFGQGTKVIEK VL10 F112Y 200EIVLTQSPGTLSLSPGERATLSCRASQGI SRSYLAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQYGSSPWTFGQGTKVEIK VL11 Y53E 202EIVLTQSPGTLSLSPGERATLSCRASQGI SRSELAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQFGSSPWTFGQGTKVEIK VL12 Y53R 204EIVLTQSPGTLSLSPGERATLSCRASQGI SRSRLAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQFGSSPWTFGQGTKVEIK VL13 F112E 206EIVLTQSPGTLSLSPGERATLSCRASQGI SRSYLAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQEGSSPWTFGQGTKVEIK VL14 F112R 208EIVLTQSPGTLSLSPGERATLSCRASQGI SRSYLAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQRGSSPWTFGQGTKVEIK VL15 Y53A, 210EIVLTQSPGTLSLSPGERATLSCRASQGI F112A SRSALAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQRGSSPWTFGQGTKVEIK VL16 G48S, I49V,212 EIVLTQSPGTLSLSPGERATLSCRASQSV R51S SSSYLAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQFGSSPWTFGQGTKVEIK

TABLE 2B Exemplary V_(H) Chains. Optional N-terminal signalsequences are not shown, but may be reflectedin the arbitrary “description” of the V_(H). SEQ Desig- ID nationDescription NO Amino Acid Sequence VH1 Anti-IL-17R 305QVQLVQSGAEVKKPGASVKVSCKASG Wild type YTFTRYGISWVRQAPGQGLEWMGWIS (WT)TYSGNTNYAQKLQGRVTMTTDTSTST AYMELRSLRSDDTAVYYCARRQLYFD YWGQGTLVTVSS VH2Y124A 307 QVQLVQSGAEVKKPGASVKVSCKASG YTFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTST AYMELRSLRSDDTAVYYCARRQLYFD AWGQGTLVTVSS VH3F122A 309 QVQLVQSGAEVKKPGASVKVSCKASG YTFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTST AYMELRSLRSDDTAVYYCARRQLYAD YWGQGTLVTVSS VH4Y121A 311 QVQLVQSGAEVKKPGASVKVSCKASG YTFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTST AYMELRSLRSDDTAVYYCARRQLAFD YWGQGTLVTVSS VH5Y79A 313 QVQLVQSGAEVKKPGASVKVSCKASG YTFTRYGISWVRQAPGQGLEWMGWISTYSGNTNAAQKLQGRVTMTTDTSTST AYMELRSLRSDDTAVYYCARRQLYFD YWGQGTLVTVSS VH6Y73A 315 QVQLVQSGAEVKKPGASVKVSCKASG YTFTRYGISWVRQAPGQGLEWMGWISTASGNTNYAQKLQGRVTMTTDTSTST AYMELRSLRSDDTAVYYCARRQLYFD YWGQGTLVTVSS VH7W69A 317 QVQLVQSGAEVKKPGASVKVSCKASG YTFTRYGISWVRQAPGQGLEWMGAISTYSGNTNYAQKLQGRVTMTTDTSTSTA YMELRSLRSDDTAVYYCARRQLYFDY WGQGTLVTVSS VH8Y51A 319 QVQLVQSGAEVKKPGASVKVSCKASG YTFTRAGISWVRQAPGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTST AYMELRSLRSDDTAVYYCARRQLYFD YWGQGTLVTVSS VH9L120Q 321 QVQLVQSGAEVKKPGASVKVSCKASG YTFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTST AYMELRSLRSDDTAVYYCARRQQYFD YWGQGTLVTVSS VH10R118A 323 QVQLVQSGAEVKKPGASVKVSCKASG YTFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTST AYMELRSLRSDDTAVYYCARAQLYFD YWGQGTLVTVSS VH11R118A,L120Q 325 QVQLVQSGAEVKKPGASVKVSCKASG YTFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTST AYMELRSLRSDDTAVYYCARAQQYFD YWGQGTLVTVSS VH12Anti-huTR2 327 QVQLQESGPGLVKPSQTLSLTCTVSGG Wild typeSISSGDYFWSWIRQLPGKGLEWIGHIHN (WT) SGTTYYNPSLKSRVTISVDTSKKQFSLRLSSVTAADTAVYYCARDRGGDYYYG MDVWGQGTTVTVSS VH13 D123A 329QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGAYYYG MDVWGQGTTVTVSSVH14 Y124A 331 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDAYYG MDVWGQGTTVTVSSVH15 Y53A 333 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDAFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDYYYG MDVWGQGTTVTVSSVH16 F54A 335 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYAWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSL RLSSVTAADTAVYYCARDRGGDYYYG MDVWGQGTTVTVSSVH17 F54E 337 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYEWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDYYYG MDVWGQGTTVTVSSVH18 F54Y 339 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYYWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSL RLSSVTAADTAVYYCARDRGGDYYYG MDVWGQGTTVTVSSVH19 F54R 341 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYRWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDYYYG MDVWGQGTTVTVSSVH20 W55A 343 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYFASWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDYYYG MDVWGQGTTVTVSSVH21 Y79A 345 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTAYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDYYYG MDVWGQGTTVTVSSVH22 Y80A 347 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYANPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDYYYG MDVWGQGTTVTVSSVH23 Y125A 349 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDYAYG MDVWGQGTTVTVSSVH24 Y126A 351 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDYYAG MDVWGQGTTVTVSSVH25 Y125E 353 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDYEYGM DVWGQGTTVTVSSVH26 Y125R 355 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDYRYGM DVWGQGTTVTVSSVH27 F54A, Y125A 357 QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYAWSWIRQLPGKGLEWIGHIH NSGTTYYNPSLKSRVTISVDTSKKQFSLRLSSVTAADTAVYYCARDRGGDYAYG MDVWGQGTTVTVSS VH28 F54A, Y126A 359QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYAWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSL RLSSVTAADTAVYYCARDRGGDYYAG MDVWGQGTTVTVSSVH29 Y126E 361 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDYYEGM DVWGQGTTVTVSSVH30 Y126R 363 QVQLQESGPGLVKPSQTLSLTCTVSGG SISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDYYRGM DVWGQGTTVTVSSVH31 F54A,  365 QVQLQESGPGLVKPSQTLSLTCTVSGG Y125A,SISSGDYAWSWIRQLPGKGLEWIGHIH Y126A NSGTTYYNPSLKSRVTISVDTSKKQFSLRLSSVTAADTAVYYCARDRGGDYAAG MDVWGQGTTVTVSS VH32 D123A, 367QVQLQESGPGLVKPSQTLSLTCTVSGG Y124A SISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGAAYYG MDVWGQGTTVTVSSVH33 Y125A, 369 QVQLQESGPGLVKPSQTLSLTCTVSGG Y126ASISSGDYFWSWIRQLPGKGLEWIGHIHN SGTTYYNPSLKSRVTISVDTSKKQFSLRLSSVTAADTAVYYCARDRGGDYAAG MDVWGQGTTVTVSS VH34 Y124A, 371QVQLQESGPGLVKPSQTLSLTCTVSGG Y125A, SISSGDYFWSWIRQLPGKGLEWIGHIHN Y126ASGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDRGGDAAAG MDVWGQGTTVTVSSVH35 H71Y, H73Y, 373 QVQLQESGPGLVKPSQTLSLTCTVSGG N74Y, T77SSISSGDYFWSWIRQLPGKGLEWIGYIYY SGSTYYNPSLKSRVTISVDTSKKQFSLRLSSVTAADTAVYYCARDRGGDYYYG MDVWGQGTTVTVSS VH36 R120Y, 375QVQLQESGPGLVKPSQTLSLTCTVSGG G122D, SISSGDYFWSWIRQLPGKGLEWIGHIHN D125YSGTTYYNPSLKSRVTISVDTSKKQFSLR LSSVTAADTAVYYCARDYGDYYYYYY GMDVWGQGTTVTVSS

TABLE 2C Embodiments of the immunoglobulins containing the indicatedV_(L) and V_(H) (or multimers thereof), as disclosed in Tables 2A and 2Babove. Antibodies 16429 and 16449, also listed here, are positivecontrol antibodies for human IL-17R and TR2, respectively. Antibody #V_(L) V_(H) 1869 VL6 VH13 1870 VL6 VH14 1910 VL7 VH12 1911 VL9 VH12 1912VL6 VH15 1913 VL6 VH16 1914 VL6 VH20 1915 VL6 VH21 1916 VL6 VH22 1919VL6 VH23 1920 VL6 VH24 1921 VL6 VH32 1922 VL6 VH34 1961 VL8 VH16 1962VL8 VH23 1963 VL8 VH24 1964 VL7 VH24 1965 VL7 VH23 1966 VL7 VH16 2281VL16 VH12 2301 VL10 VH12 2302 VL15 VH12 2303 VL11 VH12 2304 VL12 VH122305 VL13 VH12 2306 VL14 VH12 2307 VL6 VH18 2321 VL6 VH35 2322 VL6 VH362323 VL6 VH27 2324 VL6 VH28 2325 VL6 VH19 2326 VL6 VH25 2327 VL6 VH262328 VL6 VH29 2329 VL6 VH30 2330 VL6 VH33 2331 VL6 VH31 2332 VL6 VH174241 VL8 VH25 4341 VL11 VH23 10182 VL8 VH26 10183 VL12 VH23 10184 VL12VH26 10185 VL11 VH25 10186 VL7 VH28 10187 VL7 VH27 10188 VL8 VH28 10189VL8 VH27 10190 VL15 VH24 10191 VL15 VH23 10192 VL15 VH16 16429 VL1 VH116430 VL4 VH1 16433 VL5 VH1 16434 VL1 VH10 16435 VL4 VH10 16436 VL5 VH1016437 VL1 VH9 16438 VL4 VH9 16439 VL5 VH9 16440 VL1 VH11 16441 VL4 VH1116444 VL5 VH11 16449 VL6 VH12 16613 VL8 VH12 16629 VL3 VH1 16630 VL2 VH116631 VL1 VH8 16632 VL1 VH7 16633 VL1 VH6 16634 VL1 VH5 16635 VL1 VH416636 VL1 VH3 16637 VL1 VH2

TABLE 2D Embodiments of the carrier antibodies containing the indicatedV_(L) and V_(H) (or multimers thereof), as disclosed in Tables 2A and 2Babove, and a fusion partner as described in greater detail in Examples5-6 herein. Antibody # V_(L) V_(H) Fusion partner 3742 VL1 VH1 ShK(1-35,Q16K) 10162 VL4 VH10 FGF21 10163 VL11 VH23 FGF21 10164 VL11 VH23ShK(1-35, Q16K)

In some embodiments, the immunoglobulin (including antibodies andantibody fragments) can be useful as a therapeutic molecule which can beused singularly or in combination with other therapeutics to achieve thedesired effects. In such embodiments, the inventive immunoglobulin(including antibodies and antibody fragments) further comprises one totwenty-four, one to sixteen, one to eight, or one to four,pharmacologically active chemical moieties conjugated thereto, whether asmall molecule or a polypeptide. The pharmacologically active smallmolecule or polypeptide chemical moieties can be conjugated at or viathe N-terminal or C-terminal residue of the immunoglobulin monomers(e.g., LC or HC monomers), chemical reactions known in the art andfurther described herein. Alternatively encompassed by the invention, isconjugation of the pharmacologically active chemical moiety, ormoieties, at or via functional groups on one or more side chains of theamino acid residue(s) within the primary chain of the inventiveimmunoglobulin. Useful methods and internal conjugation sites (e.g.,particular cysteine residues) within immunoglobulin chains are known inthe art (e.g., Gegg et al., Modified Fc Molecules, published in WO2007/022070 and US 20070269369, which are incorporated herein byreference in their entireties).

In other embodiments of the invention, in which the pharmacologicallyactive chemical moiety is a polypeptide, a recombinant fusion proteincan be produced with the pharmacologically active polypeptide beinginserted in the primary amino acid sequence of the of the immunoglobulinheavy chain within an internal loop of the Fc domain of theimmunoglobulin heavy chain, instead of at the N- and/or C-terminus, asfurther described in the Examples herein and in the art (e.g., Gegg etal., U.S. Pat. No. 7,442,778; U.S. Pat. No. 7,655,765; U.S. Pat. No.7,655,764; U.S. Pat. No. 7,662,931; U.S. Pat. No. 7,645,861; publishedU.S. Patent Applications US 2009/0281286; and US 2009/0286964, each ofwhich are incorporated herein by reference in their entireties).

“Conjugated” means that at least two chemical moieties are covalentlylinked, or bound to each other, either directly, or optionally, via apeptidyl or non-peptidyl linker moiety that is itself covalently linkedto both of the moieties. For example, covalent linkage can be via anamino acid residue of a peptide or protein, including via an alpha aminogroup, an alpha carboxyl group, or via a side chain. The method by whichthe covalent linkage is achieved is not critical, for example, whether“conjugation” is by chemical synthetic means or by recombinantexpression of fused (i.e., conjugated) partners in a fusion protein.

As stated above, some embodiments of the inventive compositions involveat least one pharmacologically active polypeptide moiety conjugated tothe pharmacologically inactive immunoglobulin of the invention, forexample constituting a recombinant fusion protein of thepharmacologically active polypeptide moiety conjugated to thepharmacologically inactive immunoglobulin of the invention. The term“pharmacologically active” means that a substance so described isdetermined to have activity that affects a medical parameter (e.g.,blood pressure, blood cell count, cholesterol level, pain perception) ordisease state (e.g., cancer, autoimmune disorders, chronic pain),excluding mere immunogenicity, if any, of the substance. Conversely, theterm “pharmacologically inactive” means that no activity affecting amedical parameter or disease state can be determined for that substance,excluding mere immunogenicity, if any, of the substance. Thus,pharmacologically active peptides or proteins comprise agonistic ormimetic and antagonistic peptides as defined below. The presentinvention encompasses the use of any pharmacologically active protein,which has an amino acid sequence ranging from about 5 to about 80 aminoacid residues in length, and which is amenable to recombinantexpression. In some useful embodiments of the invention, thepharmacologically active protein is modified in one or more waysrelative to a native sequence of interest, including amino acidadditions or insertions, amino acid deletions, peptide truncations,amino acid substitutions, or chemical derivatization of amino acidresidues (accomplished by known chemical techniques), so long as therequisite bioactivity is maintained.

The terms “-mimetic peptide,” “peptide mimetic,” and “-agonist peptide”refer to a peptide or protein having biological activity comparable to anaturally occurring protein of interest, for example, but not limitedto, a toxin peptide molecule, e.g., ShK or OSK1 toxin peptides, orpeptide analogs thereof. These terms further include peptides thatindirectly mimic the activity of a naturally occurring peptide molecule,such as by potentiating the effects of the naturally occurring molecule.

The term “-antagonist peptide,” “peptide antagonist,” and “inhibitorpeptide” refer to a peptide that blocks or in some way interferes withthe biological activity of a receptor of interest, or has biologicalactivity comparable to a known antagonist or inhibitor of a receptor ofinterest (such as, but not limited to, an ion channel or a G-ProteinCoupled Receptor (GPCR)).

Examples of pharmacologically active proteins that can be used withinthe present invention include, but are not limited to, a toxin peptide(e.g., OSK1 or an OSK1 peptide analog; ShK or an ShK peptide analog), anIL-6 binding peptide, a CGRP peptide antagonist, a bradykinin B1receptor peptide antagonist, a parathyroid hormone (PTH) agonistpeptide, a parathyroid hormone (PTH) antagonist peptide, an ang-1binding peptide, an ang-2 binding peptide, a myostatin binding peptide,an erythropoietin-mimetic (EPO-mimetic) peptide, a FGF21 peptide, athrombopoietin-mimetic (TPO-mimetic) peptide (e.g., AMP2 or AMPS), anerve growth factor (NGF) binding peptide, a B cell activating factor(BAFF) binding peptide, and a glucagon-like peptide (GLP)-1 or a peptidemimetic therof or GLP-2 or a peptide mimetic thereof.

Glucagon-like peptide 1 (GLP-1) and the related peptide glucagon areproduced via differential processing of proglucagon and have opposingbiological activities. Proglucagon itself is produced in α-cells of thepancreas and in the enteroendocrine L-cells, which are located primarilyin the distal small intestine and colon. In the pancreas, glucagon isselectively cleaved from proglucagon. In the intestine, in contrast,proglucagon is processed to form GLP-1 and glucagon-like peptide 2(GLP-2), which correspond to amino acid residues 78-107 and 126-158 ofproglucagon, respectively (see, e.g., Irwin and Wong, 1995, Mol.Endocrinol. 9:267-277 and Bell et al., 1983, Nature 304:368-371). Byconvention, the numbering of the amino acids of GLP-1 is based on theGLP-1 (1-37) formed from cleavage of proglucagon. The biologicallyactive forms are generated from further processing of this peptide,which, in one numbering convention, yields GLP-1 (7-37)-OH and GLP-1(7-36)-NH₂. Both GLP-1 (7-37)-OH (or simply GLP-1 (7-37)) and GLP-1(7-36)-NH₂ have the same activities. For convenience, the term “GLP-1”,is used to refer to both of these forms. The first amino acid of theseprocessed peptides is His7 in this numbering convention. Anothernumbering convention recognized in the art, however, assumes that thenumbering of the processed peptide begins with His as position 1 ratherthan position 7. Thus, in this numbering scheme, GLP-1 (1-31) is thesame as GLP-1(7-37), and GLP-1(1-30) is the same as GLP-1 (7-36).Examples of GLP-1 mimetic polypeptide sequences include:

(SEQ ID NO: 290) HGEGTFTSDQSSYLEGQAAKEFIAWLVKGRG//; (SEQ ID NO: 291)HGEGTFTSDQSSYLEGQAAKEFIAWLQKGRG//; (SEQ ID NO: 292)HGEGTFTSDVSSYQEGQAAKEFIAWLVKGRG//; (SEQ ID NO: 293)HGEGTFTSDVSSYLEGQAAKEFIAQLVKGRG//; (SEQ ID NO: 294)HGEGTFTSDVSSYLEGQAAKEFIAQLQKGRG//; (SEQ ID NO: 295)HGEGTFTSDVSSYLEGQAAKEFIAWLQKGRG//; (SEQ ID NO: 296)HNETTFTSDVSSYLEGQAAKEFIAWLVKGRG// (SEQ ID NO: 297)HGEGTFTSDVSSYLENQTAKEFIAWLVKGRG//; (SEQ ID NO: 298)HGEGTFTSDVSSYLEGNATKEFIAWLVKGRG//; (SEQ ID NO: 299)HGEGTFTSDVSSYLEGQAAKEFIAWLVNGTG//; (SEQ ID NO: 300)HGEGTFTSDVSSYLEGQAAKEFIAWLVKNRT//; (SEQ ID NO: 301)HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRNGT//; (SEQ ID NO: 302)HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGTGNGT//; and (SEQ ID NO: 303)HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGNGT//.

Human GLP-2 and GLP-2-mimetic analogs are also known in the art. (See,e.g., Prasad et al., Glucagonlike peptide-2 analogue enhances intestinalmucosal mass after ischemia and reperfusion, J. Pediatr. Surg. 2000February; 35(2):357-59 (2000); Yusta et al., Glucagon-like peptide-2receptor activation engages bad and glycogen synthase kinase-3 in aprotein kinase A-dependent manner and prevents apoptosis followinginhibition of phosphatidylinositol 3-kinase, J. Biol. Chem.277(28):24896-906 (2002)).

“Toxin peptides” include peptides and polypeptides having the same aminoacid sequence of a naturally occurring pharmacologically active peptideor polypeptide that can be isolated from a venom, and also includemodified peptide analogs of such naturally occurring molecules. (See,e.g., Kalman et al., ShK-Dap22, a potent Kv1.3-specificimmunosuppressive polypeptide, J. Biol. Chem. 273(49):32697-707 (1998);Kem et al., U.S. Pat. No. 6,077,680; Mouhat et al., OsK1 derivatives, WO2006/002850 A2; Chandy et al., Analogs of SHK toxin and their uses inselective inhibition of Kv1.3 potassium channels, WO 2006/042151;Sullivan et al., Toxin Peptide therapeutic agents, WO 2006/116156 A2,all of which are incorporated herein by reference in their entirety).Snakes, scorpions, spiders, bees, snails and sea anemone are a fewexamples of organisms that produce venom that can serve as a rich sourceof small bioactive toxin peptides or “toxins” that potently andselectively target ion channels and receptors. An example of a toxinpeptide is OSK1 (also known as OsK1), a toxin peptide isolated fromOrthochirus scrobiculosus scorpion venom. (e.g., Mouhat et al., K+channel types targeted by synthetic OSK1, a toxin from Orthochirusscrobiculosus scorpion venom, Biochem. J. 385:95-104 (2005); Mouhat etal., Pharmacological profiling of Orthochirus scrobiculosus toxin 1analogs with a trimmed N-terminal domain, Molec. Pharmacol. 69:354-62(2006); Mouhat et al., OsK1 derivatives, WO 2006/002850 A2). Anotherexample is ShK, isolated from the venom of the sea anemone Stichodactylahelianthus. (E.g., Tudor et al., Ionisation behaviour and solutionproperties of the potassium-channel blocker ShK toxin, Eur. J. Biochem.251(1-2):133-41(1998); Pennington et al., Role of disulfide bonds in thestructure and potassium channel blocking activity of ShK toxin, Biochem.38(44): 14549-58 (1999); Kem et al., ShK toxin compositions and methodsof use, U.S. Pat. No. 6,077,680; Lebrun et al., Neuropeptidesoriginating in scorpion, U.S. Pat. No. 6,689,749; Beeton et al.,Targeting effector memory T cells with a selective peptide inhibitor ofKv1.3 channnels for therapy of autoimmune diseases, Molec. Pharmacol.67(4):1369-81 (2005)).

The toxin peptides are usually between about 20 and about 80 amino acidsin length, contain 2-5 disulfide linkages and form a very compactstructure. Toxin peptides (e.g., from the venom of scorpions, seaanemones and cone snails) have been isolated and characterized for theirimpact on ion channels. Such peptides appear to have evolved from arelatively small number of structural frameworks that are particularlywell suited to addressing the critical issues of potency and stability.The majority of scorpion and Conus toxin peptides, for example, contain10-40 amino acids and up to five disulfide bonds, forming extremelycompact and constrained structure (microproteins) often resistant toproteolysis. The conotoxin and scorpion toxin peptides can be dividedinto a number of superfamilies based on their disulfide connections andpeptide folds. The solution structure of many of these has beendetermined by NMR spectroscopy, illustrating their compact structure andverifying conservation of their family fold. (E.g., Tudor et al.,Ionisation behaviour and solution properties of the potassium-channelblocker ShK toxin, Eur. J. Biochem. 251(1-2):133-41(1998); Pennington etal., Role of disulfide bonds in the structure and potassium channelblocking activity of ShK toxin, Biochem. 38(44): 14549-58 (1999);Jaravine et al., Three-dimensional structure of toxin OSK1 fromOrthochirus scrobiculosus scorpion venom, Biochem. 36(6):1223-32 (1997);del Rio-Portillo et al.; NMR solution structure of Cn12, a novel peptidefrom the Mexican scorpion Centruroides noxius with a typical beta-toxinsequence but with alpha-like physiological activity, Eur. J. Biochem.271(12): 2504-16 (2004); Prochnicka-Chalufour et al., Solution structureof discrepin, a new K+-channel blocking peptide from the alpha-KTx15subfamily, Biochem. 45(6):1795-1804 (2006)). Examples ofpharmacologically active toxin peptides for which the practice of thepresent invention can be useful include, but are not limited to ShK,OSK1, charybdotoxin (ChTx), kaliotoxinl KTX1), or maurotoxin, or toxinpeptide analogs of any of these, modified from the native sequences atone or more amino acid residues. Other examples are known in the art, orcan be found in Sullivan et al., WO06116156 A2 or U.S. patentapplication Ser. No. 11/406,454 (titled: Toxin Peptide TherapeuticAgents, published as US 2007/0071764); Mouhat et al., OsK1 derivatives,WO 2006/002850 A2; Sullivan et al., U.S. patent application Ser. No.11/978,076 (titled: Conjugated Toxin Peptide Therapeutic Agents, filed25 Oct. 2007, and published as US20090291885 on Nov. 26, 2009), Sullivanet al., WO 2008/088422; Lebrun et al., U.S. Pat. No. 6,689,749, andSullivan et al., Selective and Potent Peptide Inhibitors of Kv1.3, U.S.Provisional Application No. 61/210,594, filed Mar. 20, 2009, which areeach incorporated by reference in their entireties.

The term “peptide analog” refers to a peptide having a sequence thatdiffers from a peptide sequence existing in nature by at least one aminoacid residue substitution, internal addition, or internal deletion of atleast one amino acid, and/or amino- or carboxy-terminal end truncations,or additions). An “internal deletion” refers to absence of an amino acidfrom a sequence existing in nature at a position other than the N- orC-terminus. Likewise, an “internal addition” refers to presence of anamino acid in a sequence existing in nature at a position other than theN- or C-terminus. “Toxin peptide analogs”, such as, but not limited to,an OSK1 peptide analog, ShK peptide analog, or ChTx peptide analog,contain modifications of a native toxin peptide sequence of interest(e.g., amino acid residue substitutions, internal additions orinsertions, internal deletions, and/or amino- or carboxy-terminal endtruncations, or additions as previously described above) relative to anative toxin peptide sequence of interest.

A “CGRP peptide antagonist” is a peptide that preferentially binds theCGRP₁ receptor, such as, but not limited to, a CGRP peptide analog, andthat antagonizes, blocks, decreases, reduces, impedes, or inhibits CGRP₁receptor activation by full length native human αCGRP or βCGRP underphysiological conditions of temperature, pH, and ionic strength. CGRPpeptide antagonists include full and partial antagonists. Suchantagonist activity can be detected by known in vitro methods or in vivofunctional assay methods. (See, e.g., Smith et al., Modifications to theN-terminus but not the C-terminus of calcitonin gene-relatedpeptide(8-37) produce antagonists with increased affinity, J. Med.Chem., 46:2427-2435 (2003)). Examples of useful CGRP peptide antagonistsare disclosed in Gegg et al., CGRP peptide antagonists and conjugates,WO 2007/048026 A2 and U.S. Ser. No. 11/584,177, filed on Oct. 19, 2006,published as US 2008/0020978 A1, which is incorporated herein byreference in its entirety.

The terms “parathyroid hormone (PTH) agonist” and “PTH agonist” refer toa molecule that binds to PTH-1 or PTH-2 receptor and increases ordecreases one or more PTH activity assay parameters as does full-lengthnative human parathyroid hormone. Examples of useful PTH agonistpeptides are disclosed in Table 1 of U.S. Pat. No. 6,756,480, titledModulators of receptors for parathyroid hormone and parathyroidhormone-related protein, which is incorporated herein by reference inits entirety. An exemplary PTH activity assay is disclosed in Example 1of U.S. Pat. No. 6,756,480.

The term “parathyroid hormone (PTH) antagonist” refers to a moleculethat binds to PTH-1 or PTH-2 receptor and blocks or prevents the normaleffect on those parameters by full length native human parathyroidhormone. Examples of useful PTH antagonist peptides are disclosed inTable 2 of U.S. Pat. No. 6,756,480, which is incorporated herein byreference in its entirety. An exemplary PTH activity assay is disclosedin Example 2 of U.S. Pat. No. 6,756,480.

The terms “bradykinin B1 receptor antagonist peptide” and “bradykinin B1receptor peptide antagonist” mean a peptide with antagonist activitywith respect to human bradykinin B1 receptor (hB1). Useful bradykinin B1receptor antagonist peptides can be identified or derived as describedin Ng et al., Antagonist of the bradykinin B1 receptor, US 2005/0215470A1, published Sep. 29, 2005, which issued as U.S. Pat. No. 7,605,120;U.S. Pat. Nos. 5,834,431 or 5,849,863. An exemplary B1 receptor activityassays are disclosed in Examples 6-8 of US 2005/0215470 A1.

The terms “thrombopoietin (TPO)-mimetic peptide” and “TPO-mimeticpeptide” refer to peptides that can be identified or derived asdescribed in Cwirla et al. (1997), Science 276: 1696-9, U.S. Pat. Nos.5,869,451 and 5,932,946, which are incorporated by reference in theirentireties; U.S. Pat. App. No. 2003/0176352, published Sep. 18, 2003,which is incorporated by reference in its entirety; WO 03/031589,published Apr. 17, 2003; WO 00/24770, published May 4, 2000; and anypeptides appearing in Table 5 of published application US 2006/0140934(U.S. Ser. No. 11/234,731, filed Sep. 23, 2005, titled Modified FcMolecules, which is incorporated herein by reference in its entirety).Those of ordinary skill in the art appreciate that each of thesereferences enables one to select different peptides than actuallydisclosed therein by following the disclosed procedures with differentpeptide libraries.

The terms “EPO-mimetic peptide” and “erythropoietin-mimetic peptide”refers to peptides that can be identified or derived as described inWrighton et al. (1996), Science 273: 458-63, and Naranda et al. (1999),Proc. Natl. Acad. Sci. USA 96: 7569-74, both of which are incorporatedherein by reference in their entireties. Useful EPO-mimetic peptidesinclude EPO-mimetic peptides listed in Table 5 of published U.S. patentapplication US 2007/0269369 A1 and in U.S. Pat. No. 6,660,843, which areboth hereby incorporated by reference in their entireties.

The term “ang-2-binding peptide” comprises peptides that can beidentified or derived as described in U.S. Pat. App. No. 2003/0229023,published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003; U.S.2003/0236193, published Dec. 25, 2003 (each of which is incorporatedherein by reference in its entirety); and any peptides appearing inTable 6 of published application US 2006/0140934 (U.S. Ser. No.11/234,731, filed Sep. 23, 2005, titled Modified Fc Molecules, which isincorporated herein by reference in its entirety). Those of ordinaryskill in the art appreciate that each of these references enables one toselect different peptides than actually disclosed therein by followingthe disclosed procedures with different peptide libraries.

The terms “nerve growth factor (NGF) binding peptide” and “NGF-bindingpeptide” comprise peptides that can be identified or derived asdescribed in WO 04/026329, published Apr. 1, 2004 and any peptidesidentified in Table 7 of published application US 2006/0140934 (U.S.Ser. No. 11/234,731, filed Sep. 23, 2005, titled Modified Fc Molecules,which is incorporated herein by reference in its entirety). Those ofordinary skill in the art appreciate that this reference enables one toselect different peptides than actually disclosed therein by followingthe disclosed procedures with different peptide libraries.

The term “myostatin-binding peptide” comprises peptides that can beidentified or derived as described in U.S. Ser. No. 10/742,379, filedDec. 19, 2003, which is incorporated herein by reference in itsentirety, and peptides appearing in Table 8 of published application US2006/0140934 (U.S. Ser. No. 11/234,731, filed Sep. 23, 2005, titledModified Fc Molecules, which is incorporated herein by reference in itsentirety). Those of ordinary skill in the art appreciate that each ofthese references enables one to select different peptides than actuallydisclosed therein by following the disclosed procedures with differentpeptide libraries.

The terms “BAFF-antagonist peptide” and “BAFF binding peptide” comprisepeptides that can be identified or derived as described in U.S. Pat.Appln. No. 2003/0195156 A1, which is incorporated herein by reference inits entirety and those peptides appearing in Table 9 of publishedapplication US 2006/0140934 (U.S. Ser. No. 11/234,731, filed Sep. 23,2005, titled Modified Fc Molecules, which is incorporated herein byreference in its entirety). Those of ordinary skill in the artappreciate that the foregoing references enable one to select differentpeptides than actually disclosed therein by following the disclosedprocedures with different peptide libraries.

The foregoing are intended merely as non-limiting examples of thepharmacologically active polypeptides that can be usefully conjugated orfused to the inventive immunoglobulins (including antibodies andantibody fragements). Any include pharmacologically active polypeptidemoiety can be used within the scope of the invention, including apolypeptide having a so-called avimer structure (see, e.g., Kolkman etal., Novel Proteins with Targeted Binding, US 2005/0089932; Baker etal., IL-6 Binding Proteins, US 2008/0281076; Stemmer et al., ProteinScaffolds and Uses Thereof, US 2006/0223114 and US 2006/0234299).

Useful preclinical animal models are known in the art for use invalidating a drug in a therapeutic indication of interest (e.g., anadoptive-transfer model of periodontal disease by Valverde et al., J.Bone Mineral Res. 19:155 (2004); an ultrasonic perivascular Doppler flowmeter-based animal model of arterial thrombosis in Gruner et al., Blood105:1492-99 (2005); pulmonary thromboembolism model, aorta occlusionmodel, and murine stroke model in Braun et al., WO 2009/115609 A1). Forexample, an adoptive transfer experimental autoimmune encephalomyelitis(AT-EAE) model of multiple sclerosis has been described forinvestigations concerning immune diseases, such as multiple sclerosis(Beeton et al., J. Immunol. 166:936 (2001); Beeton et al., PNAS 98:13942(2001); Sullivan et al., Example 45 of WO 2008/088422 A2, incorporatedherein by reference in its entirety). In the AT-EAE model, significantlyreduced disease severity and increased survival are expected for animalstreated with an effective amount of the inventive pharmaceuticalcomposition, while untreated animals are expected to develop severedisease and/or mortality. For running the AT-EAE model, theencephalomyelogenic CD4+ rat T cell line, PAS, specific for myelin-basicprotein (MBP) originated from Dr. Evelyne Beraud. The maintenance ofthese cells in vitro and their use in the AT-EAE model has beendescribed earlier [Beeton et al. (2001) PNAS 98, 13942]. PAS T cells aremaintained in vitro by alternating rounds of antigen stimulation oractivation with MBP and irradiated thymocytes (2 days), and propagationwith T cell growth factors (5 days). Activation of PAS T cells(3×10⁵/ml) involves incubating the cells for 2 days with 10 μg/ml MBPand 15×10⁶/ml syngeneic irradiated (3500 rad) thymocytes. On day 2 afterin vitro activation, 10-15×10⁶ viable PAS T cells are injected into 6-12week old female Lewis rats (Charles River Laboratories) by tail IV.Daily subcutaneous injections of vehicle (2% Lewis rat serum in PBS) ortest pharmaceutical composition are given from days—1 to 3, where day—1represent 1 day prior to injection of PAS T cells (day 0). In vehicletreated rats, acute EAE is expected to develop 4 to 5 days afterinjection of PAS T cells. Typically, serum is collected by tail veinbleeding at day 4 and by cardiac puncture at day 8 (end of the study)for analysis of levels of inhibitor. Rats are typically weighed ondays—1, 4, 6, and 8. Animals may be scored blinded once a day from theday of cell transfer (day 0) to day 3, and twice a day from day 4 to day8. Clinical signs are evaluated as the total score of the degree ofparesis of each limb and tail. Clinical scoring: 0=No signs, 0.5=distallimp tail, 1.0=limp tail, 2.0=mild paraparesis, ataxia, 3.0=moderateparaparesis, 3.5=one hind leg paralysis, 4.0=complete hind legparalysis, 5.0=complete hind leg paralysis and incontinence,5.5=tetraplegia, 6.0=moribund state or death. Rats reaching a score of5.0 are typically euthanized.

Production of Antibody Embodiments of the Immunoglobulins

Polyclonal Antibodies.

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. Alternatively, antigen may be injected directlyinto the animal's lymph node (see Kilpatrick et al., Hybridoma,16:381-389, 1997). An improved antibody response may be obtained byconjugating the relevant antigen to a protein that is immunogenic in thespecies to be immunized, e.g., keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctionalor derivatizing agent, for example, maleimidobenzoyl sulfosuccinimideester (conjugation through cysteine residues), N-hydroxysuccinimide(through lysine residues), glutaraldehyde, succinic anhydride or otheragents known in the art.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg of the protein or conjugate (formice) with 3 volumes of Freund's complete adjuvant and injecting thesolution intradermally at multiple sites. One month later, the animalsare boosted with ⅕ to 1/10 the original amount of peptide or conjugatein Freund's complete adjuvant by subcutaneous injection at multiplesites. At 7-14 days post-booster injection, the animals are bled and theserum is assayed for antibody titer. Animals are boosted until the titerplateaus. Preferably, the animal is boosted with the conjugate of thesame antigen, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

Monoclonal Antibodies.

The inventive immunoglobulins that are provided include monoclonalantibodies. Monoclonal antibodies may be produced using any techniqueknown in the art, e.g., by immortalizing spleen cells harvested from thetransgenic animal after completion of the immunization schedule. Thespleen cells can be immortalized using any technique known in the art,e.g., by fusing them with myeloma cells to produce hybridomas. Forexample, monoclonal antibodies may be made using the hybridoma methodfirst described by Kohler et al., Nature, 256:495 (1975), or may be madeby recombinant DNA methods from predetermined sequences as is useful inthe present invention (e.g., Cabilly et al., Methods of producingimmunoglobulins, vectors and transformed host cells for use therein,U.S. Pat. No. 6,331,415), including methods, such as the “split DHFR”method, that facilitate the generally equimolar production of light andheavy chains, optionally using mammalian cell lines (e.g., CHO cells)that can glycosylate the antibody (See, e.g., Page, Antibody production,EPO481790 A2 and U.S. Pat. No. 5,545,403).

Generally, in the hybridoma method, which is not useful in theproduction of the inventive immunoglobulins, but is useful to produceantigen binding proteins, a mouse or other appropriate host mammal, suchas rats, hamster or macaque monkey, is immunized as herein described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

In some instances, a hybridoma cell line is produced by immunizing atransgenic animal having human immunoglobulin sequences with animmunogen; harvesting spleen cells from the immunized animal; fusing theharvested spleen cells to a myeloma cell line, thereby generatinghybridoma cells; establishing hybridoma cell lines from the hybridomacells, and identifying a hybridoma cell line that produces an antibodythat binds to an tigen of interest. Such hybridoma cell lines, andmonoclonal antibodies produced by them, are aspects of the presentinvention.

The hybridoma cells, once prepared, are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Myelomacells for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas). Examples of suitable cell lines for use in mouse fusionsinclude Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5/5XXO Bul; examples of celllines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by BIAcore® or Scatchardanalysis (Munson et al., Anal. Biochem., 107:220 (1980); Fischer et al.,A peptide-immunoglobulin-conjugate, WO 2007/045463 A1, Example 10, whichis incorporated herein by reference in its entirety).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

Hybridomas or mAbs may be further screened to identify mAbs withparticular properties, such as the ability to inhibit K¹⁺ flux thoughKv1.x channels. Examples of such screens are provided in the examplesbelow. The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, affinity chromatography, or any othersuitable purification technique known in the art.

Recombinant Production of Antibodies.

The present invention provides isolated nucleic acids encoding any ofthe antibodies (polyclonal and monoclonal), including antibodyfragments, of the invention described herein, optionally operably linkedto control sequences recognized by a host cell, vectors and host cellscomprising the nucleic acids, and recombinant techniques for theproduction of the antibodies, which may comprise culturing the host cellso that the nucleic acid is expressed and, optionally, recovering theantibody from the host cell culture or culture medium. Similar materialsand methods apply to production of polypeptide-based immunoglobulins.

Relevant amino acid sequences from an immunoglobulin or polypeptide ofinterest may be determined by direct protein sequencing, and suitableencoding nucleotide sequences can be designed according to a universalcodon table. Alternatively, genomic or cDNA encoding the monoclonalantibodies may be isolated and sequenced from cells producing suchantibodies using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the monoclonal antibodies).

Cloning of DNA is carried out using standard techniques (see, e.g.,Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3,Cold Spring Harbor Press, which is incorporated herein by reference).For example, a cDNA library may be constructed by reverse transcriptionof polyA+mRNA, preferably membrane-associated mRNA, and the libraryscreened using probes specific for human immunoglobulin polypeptide genesequences. In one embodiment, however, the polymerase chain reaction(PCR) is used to amplify cDNAs (or portions of full-length cDNAs)encoding an immunoglobulin gene segment of interest (e.g., a light orheavy chain variable segment). The amplified sequences can be readilycloned into any suitable vector, e.g., expression vectors, minigenevectors, or phage display vectors. It will be appreciated that theparticular method of cloning used is not critical, so long as it ispossible to determine the sequence of some portion of the immunoglobulinpolypeptide of interest.

One source for antibody nucleic acids is a hybridoma produced byobtaining a B cell from an animal immunized with the antigen of interestand fusing it to an immortal cell. Alternatively, nucleic acid can beisolated from B cells (or whole spleen) of the immunized animal. Yetanother source of nucleic acids encoding antibodies is a library of suchnucleic acids generated, for example, through phage display technology.Polynucleotides encoding peptides of interest, e.g., variable regionpeptides with desired binding characteristics, can be identified bystandard techniques such as panning.

The sequence encoding an entire variable region of the immunoglobulinpolypeptide may be determined; however, it will sometimes be adequate tosequence only a portion of a variable region, for example, theCDR-encoding portion. Sequencing is carried out using standardtechniques (see, e.g., Sambrook et al. (1989) Molecular Cloning: ALaboratory Guide, Vols 1-3, Cold Spring Harbor Press, and Sanger, F. etal. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467, which isincorporated herein by reference). By comparing the sequence of thecloned nucleic acid with published sequences of human immunoglobulingenes and cDNAs, one of skill will readily be able to determine,depending on the region sequenced, (i) the germline segment usage of thehybridoman immunoglobulin polypeptide (including the isotype of theheavy chain) and (ii) the sequence of the heavy and light chain variableregions, including sequences resulting from N-region addition and theprocess of somatic mutation. One source of immunoglobulin gene sequenceinformation is the National Center for Biotechnology Information,National Library of Medicine, National Institutes of Health, Bethesda,Md.

Isolated DNA can be operably linked to control sequences or placed intoexpression vectors, which are then transfected into host cells that donot otherwise produce immunoglobulin protein, to direct the synthesis ofmonoclonal antibodies in the recombinant host cells. Recombinantproduction of antibodies is well known in the art.

Nucleic acid is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, operably linkedmeans that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Many vectors are known in the art. Vector components may include one ormore of the following: a signal sequence (that may, for example, directsecretion of the antibody; e.g.,ATGGACATGAGGGTGCCCGCTCAGCTCCTGGGGCTCCTGCTGCTGTGGCTGA GAGGTGCGCGCTGT//SEQ ID NO:102, which encodes the VK-1 signal peptide sequenceMDMRVPAQLLGLLLLWLRGARC// SEQ ID NO:103), an origin of replication, oneor more selective marker genes (that may, for example, confer antibioticor other drug resistance, complement auxotrophic deficiencies, or supplycritical nutrients not available in the media), an enhancer element, apromoter, and a transcription termination sequence, all of which arewell known in the art.

Cell, cell line, and cell culture are often used interchangeably and allsuch designations herein include progeny. Transformants and transformedcells include the primary subject cell and cultures derived therefromwithout regard for the number of transfers. It is also understood thatall progeny may not be precisely identical in DNA content, due todeliberate or inadvertent mutations. Mutant progeny that have the samefunction or biological activity as screened for in the originallytransformed cell are included.

Exemplary host cells include prokaryote, yeast, or higher eukaryotecells. Prokaryotic host cells include eubacteria, such as Gram-negativeor Gram-positive organisms, for example, Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacillus such as B. subtilis and B.licheniformis, Pseudomonas, and Streptomyces. Eukaryotic microbes suchas filamentous fungi or yeast are suitable cloning or expression hostsfor recombinant polypeptides or antibodies. Saccharomyces cerevisiae, orcommon baker's yeast, is the most commonly used among lower eukaryotichost microorganisms. However, a number of other genera, species, andstrains are commonly available and useful herein, such as Pichia, e.g.P. pastoris, Schizosaccharomyces pombe; Kluyveromyces, Yarrowia;Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces such asSchwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Host cells for the expression of glycosylated immunoglobulin, includingantibody, can be derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection of such cells are publicly available, e.g., theL-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyxmori NPV.

Vertebrate host cells are also suitable hosts, and recombinantproduction of antigen binding protein (including antibody) from suchcells has become routine procedure. Examples of useful mammalian hostcell lines are Chinese hamster ovary cells, including CHOK1 cells (ATCCCCL61), DXB-11, DG-44, and Chinese hamster ovary cells/-DHFR (CHO,Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, [Graham et al., J. Gen Virol. 36: 59 (1977)]; babyhamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4,Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCCCCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442);human lung cells (W138, ATCC CCL 75); human hepatoma cells (Hep G2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Matheret al., Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells or FS4cells; or mammalian myeloma cells.

Host cells are transformed or transfected with the above-describednucleic acids or vectors for production immunoglobulins and are culturedin conventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. In addition, novel vectors and transfected cell lineswith multiple copies of transcription units separated by a selectivemarker are particularly useful for the expression of immunoglobulins.

The host cells used to produce the immunoglobulins of the invention maybe cultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal.Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S.Pat. Re. No. 30,985 may be used as culture media for the host cells. Anyof these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Upon culturing the host cells, the immunoglobulin can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the immunoglobulin is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration.

The immunoglobulin (e.g., an antibody or antibody fragment) can bepurified using, for example, hydroxylapatite chromatography, cation oranion exchange chromatography, or preferably affinity chromatography,using the antigen of interest or protein A or protein G as an affinityligand. Protein A can be used to purify proteins that includepolypeptides are based on human γ1, γ2, or γ4 heavy chains (Lindmark etal., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended forall mouse isotypes and for human γ3 (Guss et al., EMBO J. 5: 15671575(1986)). The matrix to which the affinity ligand is attached is mostoften agarose, but other matrices are available. Mechanically stablematrices such as controlled pore glass or poly(styrenedivinyl)benzeneallow for faster flow rates and shorter processing times than can beachieved with agarose. Where the protein comprises a C_(H) 3 domain, theBakerbond ABX™resin (J. T. Baker, Phillipsburg, N.J.) is useful forpurification. Other techniques for protein purification such as ethanolprecipitation, Reverse Phase HPLC, chromatofocusing, SDS-PAGE, andammonium sulfate precipitation are also possible depending on theantibody to be recovered.

Chimeric, Humanized and Human Engineered™ Monoclonal Antibodies.

Chimeric monoclonal antibodies, in which the variable Ig domains of arodent monoclonal antibody are fused to human constant Ig domains, canbe generated using standard procedures known in the art (See Morrison,S. L., et al. (1984) Chimeric Human Antibody Molecules; Mouse AntigenBinding Domains with Human Constant Region Domains, Proc. Natl. Acad.Sci. USA 81, 6841-6855; and, Boulianne, G. L., et al, Nature 312,643-646. (1984)). A number of techniques have been described forhumanizing or modifying antibody sequence to be more human-like, forexample, by (1) grafting the non-human complementarity determiningregions (CDRs) onto a human framework and constant region (a processreferred to in the art as humanizing through “CDR grafting”) or (2)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like surface by replacement of surface residues (a processreferred to in the art as “veneering”) or (3) modifying selectednon-human amino acid residues to be more human, based on each residue'slikelihood of participating in antigen-binding or antibody structure andits likelihood for immunogenicity. See, e.g., Jones et al., Nature321:522 525 (1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A.,81:6851 6855 (1984); Morrison and Oi, Adv. Immunol., 44:65 92 (1988);Verhoeyer et al., Science 239:1534 1536 (1988); Padlan, Molec. Immun.28:489 498 (1991); Padlan, Molec. Immunol. 31(3):169 217 (1994); andKettleborough, C. A. et al., Protein Eng. 4(7):773 83 (1991); Co, M. S.,et al. (1994), J. Immunol. 152, 2968-2976); Studnicka et al. ProteinEngineering 7: 805-814 (1994); each of which is incorporated herein byreference in its entirety.

A number of techniques have been described for humanizing or modifyingantibody sequence to be more human-like, for example, by (1) graftingthe non-human complementarity determining regions (CDRs) onto a humanframework and constant region (a process referred to in the art ashumanizing through “CDR grafting”) or (2) transplanting the entirenon-human variable domains, but “cloaking” them with a human-likesurface by replacement of surface residues (a process referred to in theart as “veneering”) or (3) modifying selected non-human amino acidresidues to be more human, based on each residue's likelihood ofparticipating in antigen-binding or antibody structure and itslikelihood for immunogenicity. See, e.g., Jones et al., Nature 321:522525 (1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A., 81:68516855 (1984); Morrison and Oi, Adv. Immunol., 44:65 92 (1988); Verhoeyeret al., Science 239:1534 1536 (1988); Padlan, Molec. Immun. 28:489 498(1991); Padlan, Molec. Immunol. 31(3):169 217 (1994); and Kettleborough,C. A. et al., Protein Eng. 4(7):773 83 (1991); Co, M. S., et al. (1994),J. Immunol. 152, 2968-2976); Studnicka et al. Protein Engineering 7:805-814 (1994); each of which is incorporated herein by reference in itsentirety.

In one aspect of the invention, the light and heavy chain variableregions of the antibodies provided herein (see, Table 2A-B) are graftedto framework regions (FRs) from antibodies from the same, or adifferent, phylogenetic species. To create consensus human FRs, FRs fromseveral human heavy chain or light chain amino acid sequences may bealigned to identify a consensus amino acid sequence. In otherembodiments, the FRs of a heavy chain or light chain disclosed hereinare replaced with the FRs from a different heavy chain or light chain.In one aspect, rare amino acids in the FRs of the heavy and light chainsof the antibody are not replaced, while the rest of the FR amino acidsare replaced. A “rare amino acid” is a specific amino acid that is in aposition in which this particular amino acid is not usually found in anFR. Alternatively, the grafted variable regions from the one heavy orlight chain may be used with a constant region that is different fromthe constant region of that particular heavy or light chain as disclosedherein. In other embodiments, the grafted variable regions are part of asingle chain Fv antibody.

Antibodies can also be produced using transgenic animals that have noendogenous immunoglobulin production and are engineered to contain humanimmunoglobulin loci. For example, WO 98/24893 discloses transgenicanimals having a human Ig locus wherein the animals do not producefunctional endogenous immunoglobulins due to the inactivation ofendogenous heavy and light chain loci. WO 91/10741 also disclosestransgenic non-primate mammalian hosts capable of mounting an immuneresponse to an immunogen, wherein the antibodies have primate constantand/or variable regions, and wherein the endogenous immunoglobulinencoding loci are substituted or inactivated. WO 96/30498 discloses theuse of the Cre/Lox system to modify the immunoglobulin locus in amammal, such as to replace all or a portion of the constant or variableregion to form a modified antibody molecule. WO 94/02602 disclosesnon-human mammalian hosts having inactivated endogenous Ig loci andfunctional human Ig loci. U.S. Pat. No. 5,939,598 discloses methods ofmaking transgenic mice in which the mice lack endogenous heavy chains,and express an exogenous immunoglobulin locus comprising one or morexenogeneic constant regions.

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human-derived monoclonal antibodies. Immunization protocols,adjuvants, and the like are known in the art, and are used inimmunization of, for example, a transgenic mouse as described in WO96/33735. The monoclonal antibodies can be tested for the ability toinhibit or neutralize the biological activity or physiological effect ofthe corresponding protein. See also Jakobovits et al., Proc. Natl. Acad.Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggermann et al., Year in Immuno., 7:33 (1993); Mendez et al., Nat.Genet. 15:146-156 (1997); and U.S. Pat. No. 5,591,669, U.S. Pat. No.5,589,369, U.S. Pat. No. 5,545,807; and U.S Patent Application No.20020199213. U.S. Patent Application No. and 20030092125 describesmethods for biasing the immune response of an animal to the desiredepitope. Human antibodies may also be generated by in vitro activated Bcells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Antibody Production by Phage Display Techniques

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided another meansfor generating human-derived antibodies. Phage display is described ine.g., Dower et al., WO 91/17271, McCafferty et al., WO 92/01047, andCaton and Koprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454 (1990),each of which is incorporated herein by reference in its entirety. Theantibodies produced by phage technology are usually produced as antigenbinding fragments, e.g. Fv or Fab fragments, in bacteria and thus lackeffector functions. Effector functions can be introduced by one of twostrategies: The fragments can be engineered either into completeantibodies for expression in mammalian cells, or into bispecificantibody fragments with a second binding site capable of triggering aneffector function.

Typically, the Fd fragment (V_(H)-C_(H)1) and light chain (V_(L)-C_(L))of antibodies are separately cloned by PCR and recombined randomly incombinatorial phage display libraries, which can then be selected forbinding to a particular antigen. The antibody fragments are expressed onthe phage surface, and selection of Fv or Fab (and therefore the phagecontaining the DNA encoding the antibody fragment) by antigen binding isaccomplished through several rounds of antigen binding andre-amplification, a procedure termed panning Antibody fragments specificfor the antigen are enriched and finally isolated.

Phage display techniques can also be used in an approach for thehumanization of rodent monoclonal antibodies, called “guided selection”(see Jespers, L. S., et al., Bio/Technology 12, 899-903 (1994)). Forthis, the Fd fragment of the mouse monoclonal antibody can be displayedin combination with a human light chain library, and the resultinghybrid Fab library may then be selected with antigen. The mouse Fdfragment thereby provides a template to guide the selection.Subsequently, the selected human light chains are combined with a humanFd fragment library. Selection of the resulting library yields entirelyhuman Fab.

A variety of procedures have been described for deriving humanantibodies from phage-display libraries (See, for example, Hoogenboom etal., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol,222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson,T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, invitro selection and evolution of antibodies derived from phage displaylibraries has become a powerful tool (See Burton, D. R., and Barbas III,C. F., Adv. Immunol. 57, 191-280 (1994); and, Winter, G., et al., AnnuRev. Immunol. 12, 433-455 (1994); U.S. patent application no.20020004215 and WO92/01047; U.S. patent application no. 20030190317published Oct. 9, 2003 and U.S. Pat. No. 6,054,287; U.S. Pat. No.5,877,293.

Watkins, “Screening of Phage-Expressed Antibody Libraries by CaptureLift,” Methods in Molecular Biology, Antibody Phage Display: Methods andProtocols 178: 187-193, and U.S. Patent Application Publication No.20030044772 published Mar. 6, 2003 describes methods for screeningphage-expressed antibody libraries or other binding molecules by capturelift, a method involving immobilization of the candidate bindingmolecules on a solid support.

Other Embodiments of Immunoglobulins: Antibody Fragments

As noted above, antibody fragments comprise a portion of an intact fulllength antibody, preferably an antigen binding or variable region of theintact antibody, and include linear antibodies and multispecificantibodies formed from antibody fragments. Nonlimiting examples ofantibody fragments include Fab, Fab′, F(ab′)2, Fv, Fd, domain antibody(dAb), complementarity determining region (CDR) fragments, single-chainantibodies (scFv), single chain antibody fragments, maxibodies,diabodies, triabodies, tetrabodies, minibodies, linear antibodies,chelating recombinant antibodies, tribodies or bibodies, intrabodies,nanobodies, small modular immunopharmaceuticals (SMIPs), anantigen-binding-domain immunoglobulin fusion protein, a camelizedantibody, a VHH containing antibody, or muteins or derivatives thereof,and polypeptides that contain at least a portion of an immunoglobulinthat is sufficient to confer specific antigen binding to thepolypeptide, such as a CDR sequence, as long as the antibody retains thedesired biological activity. Such antigen fragments may be produced bythe modification of whole antibodies or synthesized de novo usingrecombinant DNA technologies or peptide synthesis.

Additional antibody fragments include a domain antibody (dAb) fragment(Ward et al., Nature 341:544-546, 1989) which consists of a V_(H)domain.

“Linear antibodies” comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific (Zapata etal. Protein Eng. 8:1057-62 (1995)).

A “minibody” consisting of scFv fused to CH3 via a peptide linker(hingeless) or via an IgG hinge has been described in Olafsen, et al.,Protein Eng Des Sel. 2004 April; 17(4):315-23.

The term “maxibody” refers to bivalent scFvs covalently attached to theFc region of an immunoglobulin, see, for example, Fredericks et al,Protein Engineering, Design & Selection, 17:95-106 (2004) and Powers etal., Journal of Immunological Methods, 251:123-135 (2001).

Functional heavy-chain antibodies devoid of light chains are naturallyoccurring in certain species of animals, such as nurse sharks, wobbegongsharks and Camelidae, such as camels, dromedaries, alpacas and llamas.The antigen-binding site is reduced to a single domain, the VH_(H)domain, in these animals. These antibodies form antigen-binding regionsusing only heavy chain variable region, i.e., these functionalantibodies are homodimers of heavy chains only having the structure H₂L₂(referred to as “heavy-chain antibodies” or “HCAbs”). Camelized V_(HH)reportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH2, and CH3 domains and lack a CH1 domain. Classical V_(H)-onlyfragments are difficult to produce in soluble form, but improvements insolubility and specific binding can be obtained when framework residuesare altered to be more VH_(H)-like. (See, e.g., Reichman, et al., JImmunol Methods 1999, 231:25-38.) Camelized V_(HH) domains have beenfound to bind to antigen with high affinity (Desmyter et al., J. Biol.Chem. 276:26285-90, 2001) and possess high stability in solution (Ewertet al., Biochemistry 41:3628-36, 2002). Methods for generatingantibodies having camelized heavy chains are described in, for example,in U.S. Patent Publication Nos. 2005/0136049 and 2005/0037421.Alternative scaffolds can be made from human variable-like domains thatmore closely match the shark V-NAR scaffold and may provide a frameworkfor a long penetrating loop structure.

Because the variable domain of the heavy-chain antibodies is thesmallest fully functional antigen-binding fragment with a molecular massof only 15 kDa, this entity is referred to as a nanobody(Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004). A nanobodylibrary may be generated from an immunized dromedary as described inConrath et al., (Antimicrob Agents Chemother 45: 2807-12, 2001).

Intrabodies are single chain antibodies which demonstrate intracellularexpression and can manipulate intracellular protein function (Biocca, etal., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA.101:17616-21, 2004). Intrabodies, which comprise cell signal sequenceswhich retain the antibody contruct in intracellular regions, may beproduced as described in Mhashilkar et al (EMBO J 14:1542-51, 1995) andWheeler et al. (FASEB J. 17:1733-5. 2003). Transbodies arecell-permeable antibodies in which a protein transduction domains (PTD)is fused with single chain variable fragment (scFv) antibodies Heng etal., (Med Hypotheses. 64:1105-8, 2005).

Further encompassed by the invention are antibodies that are SMIPs orbinding domain immunoglobulin fusion proteins specific for targetprotein. These constructs are single-chain polypeptides comprisingantigen binding domains fused to immunoglobulin domains necessary tocarry out antibody effector functions. See e.g., WO03/041600, U.S.Patent publication 20030133939 and US Patent Publication 20030118592.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies, but can also be produced directly byrecombinant host cells. See, for example, Better et al., Science 240:1041-1043 (1988); Skerra et al. Science 240: 1038-1041 (1988); Carter etal., Bio/Technology 10:163-167 (1992).

Other Embodiments of Immunoglobulins: Multivalent Antibodies

In some embodiments, it may be desirable to generate multivalent or evena multispecific (e.g. bispecific, trispecific, etc.) monoclonalantibody. Such antibody may have binding specificities for at least twodifferent epitopes of the target antigen, or alternatively it may bindto two different molecules, e.g. to the target antigen and to a cellsurface protein or receptor. For example, a bispecific antibody mayinclude an arm that binds to the target and another arm that binds to atriggering molecule on a leukocyte such as a T-cell receptor molecule(e.g., CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI(CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defensemechanisms to the target-expressing cell. As another example, bispecificantibodies may be used to localize cytotoxic agents to cells whichexpress target antigen. These antibodies possess a target-binding armand an arm which binds the cytotoxic agent (e.g., saporin,anti-interferon-60, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Multispecific antibodies can be prepared asfull length antibodies or antibody fragments.

Additionally, the immunoglobulins (e.g., antibodies and antibodyfragments) and conjugates of the present invention can also beconstructed to fold into multivalent forms, which may improve half-lifein blood. Multivalent forms can be prepared by techniques known in theart.

Bispecific or multispecific antibodies include cross-linked or“heteroconjugate” antibodies. For example, one of the antibodies in theheteroconjugate can be coupled to avidin, the other to biotin.Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques. Another method is designed to maketetramers by adding a streptavidin-coding sequence at the C-terminus ofthe scFv. Streptavidin is composed of four subunits, so when thescFv-streptavidin is folded, four subunits associate to form a tetramer(Kipriyanov et al., Hum Antibodies Hybridomas 6(3): 93-101 (1995), thedisclosure of which is incorporated herein by reference in itsentirety).

According to another approach for making bispecific antibodies, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. One interface comprises at least a part of theC_(H)3 domain of an antibody constant domain. In this method, one ormore small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g., tyrosineor tryptophan). Compensatory “cavities” of identical or similar size tothe large side chain(s) are created on the interface of the secondantibody molecule by replacing large amino acid side chains with smallerones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers. See WO 96/27011 published Sep. 6, 1996.

Techniques for generating bispecific or multispecific antibodies fromantibody fragments have also been described in the literature. Forexample, bispecific or trispecific antibodies can be prepared usingchemical linkage. Brennan et al., Science 229:81 (1985) describe aprocedure wherein intact antibodies are proteolytically cleaved togenerate F(ab′)₂ fragments. These fragments are reduced in the presenceof the dithiol complexing agent sodium arsenite to stabilize vicinaldithiols and prevent intermolecular disulfide formation. The Fab′fragments generated are then converted to thionitrobenzoate (TNB)derivatives. One of the Fab′-TNB derivatives is then reconverted to theFab′-thiol by reduction with mercaptoethylamine and is mixed with anequimolar amount of the other Fab′-TNB derivative to form the bispecificantibody. The bispecific antibodies produced can be used as agents forthe selective immobilization of enzymes. Better et al., Science 240:1041-1043 (1988) disclose secretion of functional antibody fragmentsfrom bacteria (see, e.g., Better et al., Skerra et al. Science 240:1038-1041 (1988)). For example, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form bispecificantibodies (Carter et al., Bio/Technology 10:163-167 (1992); Shalaby etal., J. Exp. Med. 175:217-225 (1992)).

Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the productionof a fully humanized bispecific antibody F(ab′)₂ molecule. Each Fab′fragment was separately secreted from E. coli and subjected to directedchemical coupling in vitro to form the bispecfic antibody.

Various techniques for making and isolating bispecific or multispecificantibody fragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers, e.g. GCN4. (See generally Kostelny et al., J. Immunol.148(5):1547-1553 (1992).) The leucine zipper peptides from the Fos andJun proteins were linked to the Fab′ portions of two differentantibodies by gene fusion. The antibody homodimers were reduced at thehinge region to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers.

Diabodies, described above, are one example of a bispecific antibody.See, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA,90:6444-6448 (1993). Bivalent diabodies can be stabilized by disulfidelinkage.

Stable monospecific or bispecific Fv tetramers can also be generated bynoncovalent association in (scFv₂)₂ configuration or as bis-tetrabodies.Alternatively, two different scFvs can be joined in tandem to form abis-scFv.

Another strategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol. 152: 5368 (1994). One approach has been to link two scFvantibodies with linkers or disulfide bonds (Mallender and Voss, J. Biol.Chem. 269:199-2061994, WO 94/13806, and U.S. Pat. No. 5,989,830, thedisclosures of which are incorporated herein by reference in theirentireties).

Alternatively, the bispecific antibody may be a “linear antibody”produced as described in Zapata et al. Protein Eng. 8(10):1057-1062(1995). Briefly, these antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared. (Tutt et al., J.Immunol. 147:60 (1991)).

A “chelating recombinant antibody” is a bispecific antibody thatrecognizes adjacent and non-overlapping epitopes of the target antigen,and is flexible enough to bind to both epitopes simultaneously (Neri etal., J Mol Biol. 246:367-73, 1995).

Production of bispecific Fab-scFv (“bibody”) and trispecificFab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (J Immunol.165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt TechnolBiomed Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH—CH₁ (Fd)chains, e.g., to produce a tribody two scFvs are fused to C-term of Fabwhile in a bibody one scFv is fused to C-term of Fab.

In yet another method, dimers, trimers, and tetramers are produced aftera free cysteine is introduced in the parental protein. A peptide-basedcross linker with variable numbers (two to four) of maleimide groups wasused to cross link the protein of interest to the free cysteines(Cochran et al., Immunity 12(3): 241-50 (2000), the disclosure of whichis incorporated herein in its entirety).

Other Embodiments of Immunoglobulins

Inventive immunoglobulins also include peptibodies. The term “peptibody”refers to a molecule comprising an antibody Fc domain attached to atleast one peptide. The production of peptibodies is generally describedin PCT publication WO 00/24782, published May 4, 2000. Any of thesepeptides may be linked in tandem (i.e., sequentially), with or withoutlinkers. Peptides containing a cysteinyl residue may be cross-linkedwith another Cys-containing peptide, either or both of which may belinked to a vehicle. Any peptide having more than one Cys residue mayform an intrapeptide disulfide bond, as well. Any of these peptides maybe derivatized, for example the carboxyl terminus may be capped with anamino group, cysteines may be cappe, or amino acid residues maysubstituted by moieties other than amino acid residues (see, e.g.,Bhatnagar et al., J. Med. Chem. 39: 3814-9 (1996), and Cuthbertson etal., J. Med. Chem. 40: 2876-82 (1997), which are incorporated byreference herein in their entirety). The peptide sequences may beoptimized, analogous to affinity maturation for antibodies, or otherwisealtered by alanine scanning or random or directed mutagenesis followedby screening to identify the best binders. Lowman, Ann. Rev. Biophys.Biomol. Struct. 26: 401-24 (1997). Various molecules can be insertedinto the immunoglobulin structure, e.g., within the peptide portionitself or between the peptide and vehicle portions of theimmunoglobulins, while retaining the desired activity of immunoglobulin.One can readily insert, for example, molecules such as an Fc domain orfragment thereof, polyethylene glycol or other related molecules such asdextran, a fatty acid, a lipid, a cholesterol group, a smallcarbohydrate, a peptide, a detectable moiety as described herein(including fluorescent agents, radiolabels such as radioisotopes), anoligosaccharide, oligonucleotide, a polynucleotide, interference (orother) RNA, enzymes, hormones, or the like. Other molecules suitable forinsertion in this fashion will be appreciated by those skilled in theart, and are encompassed within the scope of the invention. Thisincludes insertion of, for example, a desired molecule in between twoconsecutive amino acids, optionally joined by a suitable linker.

Linkers.

A “linker” or “linker moiety”, as used interchangeably herein, refers toa biologically acceptable peptidyl or non-peptidyl organic group that iscovalently bound to an amino acid residue of a polypeptide chain (e.g.,an immunoglobulin HC or immunoglobulin LC or immunoglobulin Fc domain)contained in the inventive composition, which linker moiety covalentlyjoins or conjugates the polypeptide chain to another peptide orpolypeptide chain in the molecule, or to a therapeutic moiety, such as abiologically active small molecule or oligopeptide, or to a half-lifeextending moiety, e.g., see, Sullivan et al., Toxin Peptide TherapeuticAgents, US2007/0071764; Sullivan et al., Toxin Peptide TherapeuticAgents, PCT/US2007/022831, published as WO 2008/088422; and U.S.Provisional Application Ser. No. 61/210,594, filed Mar. 20, 2009, whichare all incorporated herein by reference in their entireties.

The presence of any linker moiety in the immunoglobulins of the presentinvention is optional. When present, the linker's chemical structure isnot critical, since it serves primarily as a spacer to position, join,connect, or optimize presentation or position of one functional moietyin relation to one or more other functional moieties of a molecule ofthe inventive immunoglobulin. The presence of a linker moiety can beuseful in optimizing pharamcologial activity of some embodiments of theinventive immunoglobulin (including antibodies and antibody fragments).The linker is preferably made up of amino acids linked together bypeptide bonds. The linker moiety, if present, can be independently thesame or different from any other linker, or linkers, that may be presentin the inventive immunoglobulin.

As stated above, the linker moiety, if present (whether within theprimary amino acid sequence of the immunoglobulin, or as a linker forattaching a therapeutic moiety or half-life extending moiety to theinventive immunoglobulin), can be “peptidyl” in nature (i.e., made up ofamino acids linked together by peptide bonds) and made up in length,preferably, of from 1 up to about 40 amino acid residues, morepreferably, of from 1 up to about 20 amino acid residues, and mostpreferably of from 1 to about 10 amino acid residues. Preferably, butnot necessarily, the amino acid residues in the linker are from amongthe twenty canonical amino acids, more preferably, cysteine, glycine,alanine, proline, asparagine, glutamine, and/or serine. Even morepreferably, a peptidyl linker is made up of a majority of amino acidsthat are sterically unhindered, such as glycine, serine, and alaninelinked by a peptide bond. It is also desirable that, if present, apeptidyl linker be selected that avoids rapid proteolytic turnover incirculation in vivo. Some of these amino acids may be glycosylated, asis well understood by those in the art. For example, a useful linkersequence constituting a sialylation site is X₁X₂NX₄X₅G (SEQ ID NO:148),wherein X₁, X₂, X₄ and X₅ are each independently any amino acid residue.

In other embodiments, the 1 to 40 amino acids of the peptidyl linkermoiety are selected from glycine, alanine, proline, asparagine,glutamine, and lysine. Preferably, a linker is made up of a majority ofamino acids that are sterically unhindered, such as glycine and alanineThus, preferred linkers include polyglycines, polyserines, andpolyalanines, or combinations of any of these. Some exemplary peptidyllinkers are poly(Gly)₁₋₈, particularly (Gly)₃, (Gly)₄ (SEQ ID NO:149),(Gly)₅ (SEQ ID NO:150) and (Gly) (SEQ ID NO:151), as well as,poly(Gly)₄Ser (SEQ ID NO:152), poly(Gly-Ala)₂₋₄ and poly(Ala)₁₋₈. Otherspecific examples of peptidyl linkers include (Gly)₅Lys (SEQ ID NO:154),and (Gly)₅LysArg (SEQ ID NO:155). Other examples of useful peptidyllinkers are: Other examples of useful peptidyl linkers are:

(Gly)₃Lys(Gly)₄; (SEQ ID NO: 159) (Gly)₃AsnGlySer(Gly)₂;(SEQ ID NO: 156) (Gly)₃Cys(Gly)₄; (SEQ ID NO: 157) and GlyProAsnGlyGly.(SEQ ID NO: 158)

To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄ meansGly-Gly-Gly-Lys-Gly-Gly-Gly-Gly (SEQ ID NO:159). Other combinations ofGly and Ala are also useful.

Commonly used linkers include those which may be identified herein as“L5” (GGGGS; or “G₄S”; SEQ ID NO:152), “L10” (GGGGSGGGGS; SEQ IDNO:153), “L25” (GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO:146) and anylinkers used in the working examples hereinafter.

In some embodiments of the compositions of this invention, whichcomprise a peptide linker moiety, acidic residues, for example,glutamate or aspartate residues, are placed in the amino acid sequenceof the linker moiety. Examples include the following peptide linkersequences:

GGEGGG; (SEQ ID NO: 160) GGEEEGGG; (SEQ ID NO: 161) GEEEG;(SEQ ID NO: 162) GEEE; (SEQ ID NO: 163) GGDGGG; (SEQ ID NO: 164)GGDDDGG; (SEQ ID NO: 165) GDDDG; (SEQ ID NO: 166) GDDD; (SEQ ID NO: 167)GGGGSDDSDEGSDGEDGGGGS; (SEQ ID NO: 168) WEWEW; (SEQ ID NO: 169) FEFEF;(SEQ ID NO: 170) EEEWWW; (SEQ ID NO: 171) EEEFFF; (SEQ ID NO: 172)WWEEEWW; (SEQ ID NO: 173) or FFEEEFF. (SEQ ID NO: 174)

In other embodiments, the linker constitutes a phosphorylation site,e.g., X₁X₂YX₄X₅G (SEQ ID NO:175), wherein X₁, X₂, X₄, and X₅ are eachindependently any amino acid residue; X₁X₂SX₄X₅G (SEQ ID NO:176),wherein X₁, X₂, X₄ and X₅ are each independently any amino acid residue;or X₁X₂TX₄X₅G (SEQ ID NO:177), wherein X₁, X₂, X₄ and X₅ are eachindependently any amino acid residue.

The linkers shown here are exemplary; peptidyl linkers within the scopeof this invention may be much longer and may include other residues. Apeptidyl linker can contain, e.g., a cysteine, another thiol, ornucleophile for conjugation with a half-life extending moiety. Inanother embodiment, the linker contains a cysteine or homocysteineresidue, or other 2-amino-ethanethiol or 3-amino-propanethiol moiety forconjugation to maleimide, iodoacetaamide or thioester, functionalizedhalf-life extending moiety.

Another useful peptidyl linker is a large, flexible linker comprising arandom Gly/Ser/Thr sequence, for example: GSGSATGGSGSTASSGSGSATH (SEQ IDNO:178) or HGSGSATGGSGSTASSGSGSAT (SEQ ID NO:179), that is estimated tobe about the size of a 1 kDa PEG molecule. Alternatively, a usefulpeptidyl linker may be comprised of amino acid sequences known in theart to form rigid helical structures (e.g., Rigid linker:-AEAAAKEAAAKEAAAKAGG-) (SEQ ID NO:180). Additionally, a peptidyl linkercan also comprise a non-peptidyl segment such as a 6 carbon aliphaticmolecule of the formula —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—. The peptidyl linkerscan be altered to form derivatives as described herein.

Optionally, a non-peptidyl linker moiety is also useful for conjugatingthe half-life extending moiety to the peptide portion of the half-lifeextending moiety-conjugated toxin peptide analog. For example, alkyllinkers such as —NH—(CH₂)_(s)—C(O)—, wherein s=2-20 can be used. Thesealkyl linkers may further be substituted by any non-sterically hinderinggroup such as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl,Br), CN, NH₂, phenyl, etc. Exemplary non-peptidyl linkers arepolyethylene glycol (PEG) linkers (e.g., shown below):

Wherein n is such that the linkers has a molecular weight of about 100to about 5000 Daltons (Da), preferably about 100 to about 500 Da.

In one embodiment, the non-peptidyl linker is aryl. The linkers may bealtered to form derivatives in the same manner as described in the art,e.g., in Sullivan et al., Toxin Peptide Therapeutic Agents,US2007/0071764; Sullivan et al., Toxin Peptide Therapeutic Agents,PCT/US2007/022831, published as WO 2008/088422; and U.S. ProvisionalApplication Ser. No. 61/210,594, filed Mar. 20, 2009, which are allincorporated herein by reference in their entireties.

In addition, PEG moieties may be attached to the N-terminal amine orselected side chain amines by either reductive alkylation using PEGaldehydes or acylation using hydroxysuccinimido or carbonate esters ofPEG, or by thiol conjugation.

“Aryl” is phenyl or phenyl vicinally-fused with a saturated,partially-saturated, or unsaturated 3-, 4-, or 5 membered carbon bridge,the phenyl or bridge being substituted by 0, 1, 2 or 3 substituentsselected from C₁₋₈ alkyl, C₁₋₄ haloalkyl or halo.

“Heteroaryl” is an unsaturated 5, 6 or 7 membered monocyclic orpartially-saturated or unsaturated 6-, 7-, 8-, 9-, 10- or 11 memberedbicyclic ring, wherein at least one ring is unsaturated, the monocyclicand the bicyclic rings containing 1, 2, 3 or 4 atoms selected from N, Oand S, wherein the ring is substituted by 0, 1, 2 or 3 substituentsselected from C₁₋₈ alkyl, C₁₋₄ haloalkyl and halo.

Non-peptide portions of the inventive composition of matter, such asnon-peptidyl linkers or non-peptide half-life extending moieties can besynthesized by conventional organic chemistry reactions.

The above is merely illustrative and not an exhaustive treatment of thekinds of linkers that can optionally be employed in accordance with thepresent invention.

Production of Immunoglobulin Variants.

As noted above, recombinant DNA- and/or RNA-mediated protein expressionand protein engineering techniques, or any other methods of preparingpeptides, are applicable to the making of the inventive compositions.For example, polypeptides can be made in transformed host cells.Briefly, a recombinant DNA molecule, or construct, coding for thepeptide is prepared. Methods of preparing such DNA molecules are wellknown in the art. For instance, sequences encoding the peptides can beexcised from DNA using suitable restriction enzymes. Any of a largenumber of available and well-known host cells may be used in thepractice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts may be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial host cells in culture include bacteria(such as Escherichia coli sp.), yeast (such as Saccharomyces sp.) andother fungal cells, insect cells, plant cells, mammalian (includinghuman) cells, e.g., CHO cells and HEK-293 cells, and others noted hereinor otherwise known in the art. Modifications can be made at the DNAlevel, as well. The peptide-encoding DNA sequence may be changed tocodons more compatible with the chosen host cell. For E. coli, optimizedcodons are known in the art. Codons can be substituted to eliminaterestriction sites or to include silent restriction sites, which may aidin processing of the DNA in the selected host cell. Next, thetransformed host is cultured and purified. Host cells may be culturedunder conventional fermentation conditions so that the desired compoundsare expressed. Such fermentation conditions are well known in the art.In addition, the DNA optionally further encodes, 5′ to the coding regionof a fusion protein, a signal peptide sequence (e.g., a secretory signalpeptide) operably linked to the expressed immunoglobulin. For furtherexamples of appropriate recombinant methods and exemplary DNA constructsuseful for recombinant expression of the inventive compositions bymammalian cells, including dimeric Fc fusion proteins (“peptibodies”) orchimeric immunoglobulin (light chain+heavy chain)-Fc heterotrimers(“hemibodies”), conjugated to specific binding agents of the invention,see, e.g., Sullivan et al., Toxin Peptide Therapeutic Agents,US2007/0071764; Sullivan et al., Toxin Peptide Therapeutic Agents,PCT/US2007/022831, published as WO 2008/088422; and U.S. ProvisionalApplication Ser. No. 61/210,594, filed Mar. 20, 2009, which are allincorporated herein by reference in their entireties.

Amino acid sequence variants of the desired immunoglobulin may beprepared by introducing appropriate nucleotide changes into the encodingDNA, or by peptide synthesis. Such variants include, for example,deletions and/or insertions and/or substitutions of residues within theamino acid sequences of the immunoglobulins or antibodies. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the immunoglobulin, such as changing thenumber or position of glycosylation sites. In certain instances,immunoglobulin variants are prepared with the intent to modify thoseamino acid residues which are directly involved in epitope binding. Inother embodiments, modification of residues which are not directlyinvolved in epitope binding or residues not involved in epitope bindingin any way, is desirable, for purposes discussed herein. Mutagenesiswithin any of the CDR regions and/or framework regions is contemplated.Covariance analysis techniques can be employed by the skilled artisan todesign useful modifications in the amino acid sequence of theimmunoglobulin, including an antibody or antibody fragment. (E.g.,Choulier, et al., Covariance Analysis of Protein Families: The Case ofthe Variable Domains of Antibodies, Proteins: Structure, Function, andGenetics 41:475-484 (2000); Demarest et al., Optimization of theAntibody C_(H)3 Domain by Residue Frequency Analysis of IgG Sequences,J. Mol. Biol. 335:41-48 (2004); Hugo et al., VL position 34 is a keydeterminant for the engineering of stable antibodies with fastdissociation rates, Protein Engineering 16(5):381-86 (2003); Aurora etal., Sequence covariance networks, methods and uses thereof, US2008/0318207 A1; Glaser et al., Stabilized polypeptide compositions, US2009/0048122 A1; Urech et al., Sequence based engineering andoptimization of single chain antibodies, WO 2008/110348 A1; Borras etal., Methods of modifying antibodies, and modified antibodies withimproved functional properties, WO 2009/000099 A2). Such modificationsdetermined by covariance analysis can improve potency, pharmacokinetic,pharmacodynamic, and/or manufacturability characteristics of animmunoglobulin.

Nucleic acid molecules encoding amino acid sequence variants of theimmunoglobulin or antibody are prepared by a variety of methods known inthe art. Such methods include oligonucleotide-mediated (orsite-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis ofan earlier prepared variant or a non-variant version of theimmunoglobulin.

Substitutional mutagenesis within any of the hypervariable or CDRregions or framework regions is contemplated. A useful method foridentification of certain residues or regions of the immunoglobulin thatare preferred locations for mutagenesis is called “alanine scanningmutagenesis,” as described by Cunningham and Wells Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed variants arescreened for the desired activity.

Some embodiments of the immunoglobulins of the present invention canalso be made by synthetic methods. Solid phase synthesis is thepreferred technique of making individual peptides since it is the mostcost-effective method of making small peptides. For example, well knownsolid phase synthesis techniques include the use of protecting groups,linkers, and solid phase supports, as well as specific protection anddeprotection reaction conditions, linker cleavage conditions, use ofscavengers, and other aspects of solid phase peptide synthesis. Suitabletechniques are well known in the art. (E.g., Merrifield (1973), Chem.Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield(1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl.10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis;U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2:105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527;“Protecting Groups in Organic Synthesis,” 3rd Edition, T. W. Greene andP. G. M. Wuts, Eds., John Wiley & Sons, Inc., 1999; NovaBiochem Catalog,2000; “Synthetic Peptides, A User's Guide,” G. A. Grant, Ed., W.H.Freeman & Company, New York, N.Y., 1992; “Advanced Chemtech Handbook ofCombinatorial & Solid Phase Organic Chemistry,” W. D. Bennet, J. W.Christensen, L. K. Hamaker, M. L. Peterson, M. R. Rhodes, and H. H.Saneii, Eds., Advanced Chemtech, 1998; “Principles of Peptide Synthesis,2nd ed.,” M. Bodanszky, Ed., Springer-Verlag, 1993; “The Practice ofPeptide Synthesis, 2nd ed.,” M. Bodanszky and A. Bodanszky, Eds.,Springer-Verlag, 1994; “Protecting Groups,” P. J. Kocienski, Ed., GeorgThieme Verlag, Stuttgart, Germany, 1994; “Fmoc Solid Phase PeptideSynthesis, A Practical Approach,” W. C. Chan and P. D. White, Eds.,Oxford Press, 2000, G. B. Fields et al., Synthetic Peptides: A User'sGuide, 1990, 77-183). For further examples of synthetic and purificationmethods known in the art, which are applicable to making the inventivecompositions of matter, see, e.g., Sullivan et al., Toxin PeptideTherapeutic Agents, US2007/0071764 and Sullivan et al., Toxin PeptideTherapeutic Agents, PCT/US2007/022831, published as WO 2008/088422 A2,which are both incorporated herein by reference in their entireties.

In further describing any of the immunoglobulins herein, as well asvariants, a one-letter abbreviation system is frequently applied todesignate the identities of the twenty “canonical” amino acid residuesgenerally incorporated into naturally occurring peptides and proteins(Table 3). Such one-letter abbreviations are entirely interchangeable inmeaning with three-letter abbreviations, or non-abbreviated amino acidnames. Within the one-letter abbreviation system used herein, an uppercase letter indicates a L-amino acid, and a lower case letter indicatesa D-amino acid. For example, the abbreviation “R” designates L-arginineand the abbreviation “r” designates D-arginine.

TABLE 3 One-letter abbreviations for the canonical amino acids.Three-letter abbreviations are in parentheses. Alanine (Ala) A Glutamine(Gln) Q Leucine (Leu) L Serine (Ser) S Arginine (Arg) R Glutamic Acid(Glu) E Lysine (Lys) K Threonine (Thr) T Asparagine (Asn) N Glycine(Gly) G Methionine (Met) M Tryptophan (Trp) W Aspartic Acid (Asp) DHistidine (His) H Phenylalanine (Phe) F Tyrosine (Tyr) Y Cysteine (Cys)C Isoleucine (Ile) I Proline (Pro) P Valine (Val) V

An amino acid substitution in an amino acid sequence is typicallydesignated herein with a one-letter abbreviation for the amino acidresidue in a particular position, followed by the numerical amino acidposition relative to an original sequence of interest, which is thenfollowed by the one-letter symbol for the amino acid residue substitutedin. For example, “T30D” symbolizes a substitution of a threonine residueby an aspartate residue at amino acid position 30, relative to theoriginal sequence of interest. Another example, “W101F” symbolizes asubstitution of a tryptophan residue by a phenylalanine residue at aminoacid position 101, relative to the original sequence of interest.

Non-canonical amino acid residues can be incorporated into a polypeptidewithin the scope of the invention by employing known techniques ofprotein engineering that use recombinantly expressing cells. (See, e.g.,Link et al., Non-canonical amino acids in protein engineering, CurrentOpinion in Biotechnology, 14(6):603-609 (2003)). The term “non-canonicalamino acid residue” refers to amino acid residues in D- or L-form thatare not among the 20 canonical amino acids generally incorporated intonaturally occurring proteins, for example, β-amino acids, homoaminoacids, cyclic amino acids and amino acids with derivatized side chains.Examples include (in the L-form or D-form) β-alanine, β-aminopropionicacid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid,aminopimelic acid, desmosine, diaminopimelic acid, N^(α)-ethylglycine,N^(α)-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine,allo-isoleucine, ω-methylarginine, N^(α)-methylglycine,N^(α)-methylisoleucine, N^(α)-methylvaline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, 0-phosphoserine,N^(α)-acetylserine, N^(α)-formylmethionine, 3-methylhistidine,5-hydroxylysine, and other similar amino acids, and those listed inTable 4 below, and derivatized forms of any of these as describedherein. Table 4 contains some exemplary non-canonical amino acidresidues that are useful in accordance with the present invention andassociated abbreviations as typically used herein, although the skilledpractitioner will understand that different abbreviations andnomenclatures may be applicable to the same substance and appearinterchangeably herein.

TABLE 4 Useful non-canonical amino acids for amino acid addition,insertion, or substitution into peptide sequences in accordance with thepresent invention. In the event an abbreviation listed in Table 4differs from another abbreviation for the same substance disclosedelsewhere herein, both abbreviations are understood to be applicable.The amino acids listed in Table 4 can be in the L-form or D-form. AminoAcid Abbreviation(s) Acetamidomethyl Acm Acetylarginine acetylargα-aminoadipic acid Aad aminobutyric acid Abu 6-aminohexanoic acid Ahx;εAhx 3-amino-6-hydroxy-2-piperidone Ahp 2-aminoindane-2-carboxylic acidAic α-amino-isobutyric acid Aib 3-amino-2-naphthoic acid Anc2-aminotetraline-2-carboxylic acid Atc Aminophenylalanine Aminophe;Amino-Phe 4-amino-phenylalanine 4AmP 4-amidino-phenylalanine 4AmPhe2-amino-2-(1- 4AmPig carbamimidoylpiperidin-4-yl)acetic acid Argψ(CH₂NH) -reduced amide bond rArg β-homoarginine bhArg β-homolysinebhomoK β-homo Tic BhTic β-homophenylalanine BhPhe β-homoproline BhProβ-homotryptophan BhTrp 4,4′-biphenylalanine Bip β,β-diphenyl-alanineBiPhA β-phenylalanine BPhe p-carboxyl-phenylalanine Cpa Citrulline CitCyclohexylalanine Cha Cyclohexylglycine Chg Cyclopentylglycine Cpg2-amino-3-guanidinopropanoic acid 3G-Dpr α,γ-diaminobutyric acid Dab2,4-diaminobutyric acid Dbu diaminopropionic acid Dapα,β-diaminopropionoic acid (or 2,3- Dpr diaminopropionic acid3,3-diphenylalanine Dip 4-guanidino phenylalanine Guf 4-guanidinoproline 4GuaPr Homoarginine hArg; hR Homocitrulline hCit HomoglutaminehQ Homolysine hLys; hK; homoLys Homophenylalanine hPhe; homoPhe4-hydroxyproline (or hydroxyproline) Hyp 2-indanylglycine (orindanylglycine) IgI indoline-2-carboxylic acid Idc Iodotyrosine I-TyrLys ψ(CH₂NH)-reduced amide bond rLys methinine oxide Met[O] methioninesulfone Met[O]₂ N^(α)-methylarginine NMeR Nα-[(CH₂)₃NHCH(NH)NH₂] N-Argsubstituted glycine N^(α)-methylcitrulline NMeCit N^(α)-methylglutamineNMeQ N^(α)-methylhomocitrulline N^(α)-MeHoCit N^(α)-methylhomolysineNMeHoK N^(α)-methylleucine N^(α)-MeL; NMeL; NMeLeu; NMe-LeuN^(α)-methyllysine NMe-Lys Nε-methyl-lysine N-eMe-K Nε-ethyl-lysineN-eEt-K Nε-isopropyl-lysine N-eIPr-K N^(α)-methylnorleucine NMeNle;NMe-Nle N^(α)-methylornithine N^(α)-MeOrn; NMeOrnN^(α)-methylphenylalanine NMe-Phe 4-methyl-phenylalanine MePheα-methylphenyalanine AMeF N^(α)-methylthreonine NMe-Thr; NMeThrN^(α)-methylvaline NMeVal; NMe-Val Nε-(O-(aminoethyl)-O′-(2-propanoyl)-K(NPeg11) undecaethyleneglycol)-LysineNε-(O-(aminoethyl)-O′-(2-propanoyl)- K(NPeg27) (ethyleneglycol)27-Lysine3-(1-naphthyl)alanine 1-Nal; 1Nal 3-(2-naphthyl)alanine 2-Nal; 2Nalnipecotic acid Nip Nitrophenylalanine nitrophe norleucine Nle norvalineNva or Nvl O-methyltyrosine Ome-Tyr octahydroindole-2-carboxylic acidOic Ornithine Orn Orn ψ(CH2NH)-reduced amide bond rOrn4-piperidinylalanine 4PipA 4-pyridinylalanine 4Pal 3-pyridinylalanine3Pal 2-pyridinylalanine 2Pal para-aminophenylalanine 4AmP; 4-Amino-Phepara-iodophenylalanine (or 4- pI-Phe iodophenylalanine) PhenylglycinePhg 4-phenyl-phenylalanine (or 4Bip biphenylalanine) 4,4′-biphenylalanine Bip pipecolic acid Pip 4-amino-1-piperidine-4-carboxylic 4Pipacid Sarcosine Sar 1,2,3,4-tetrahydroisoquinoline Tic1,2,3,4-tetrahydroisoquinoline-1- Tiq carboxylic acid1,2,3,4-tetrahydroisoquinoline-7- Hydroxyl-Tic hydroxy-3-carboxylic acid1,2,3,4-tetrahydronorharman-3- Tpi carboxylic acidthiazolidine-4-carboxylic acid Thz 3-thienylalanine Thi

Nomenclature and Symbolism for Amino Acids and Peptides by the UPAC-IUBJoint Commission on Biochemical Nomenclature (JCBN) have been publishedin the following documents: Biochem. J., 1984, 219, 345-373; Eur. J.Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2; Internat. J.Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem., 1985, 260,14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids and Peptides,1985, 16, 387-410; Biochemical Nomenclature and Related Documents, 2ndedition, Portland Press, 1992, pages 39-69.

The one or more useful modifications to peptide domains of the inventiveimmunoglobulin can include amino acid additions or insertions, aminoacid deletions, peptide truncations, amino acid substitutions, and/orchemical derivatization of amino acid residues, accomplished by knownchemical techniques. For example, the thusly modified amino acidsequence includes at least one amino acid residue inserted orsubstituted therein, relative to the amino acid sequence of the nativesequence of interest, in which the inserted or substituted amino acidresidue has a side chain comprising a nucleophilic or electrophilicreactive functional group by which the peptide is conjugated to a linkerand/or half-life extending moiety. In accordance with the invention,useful examples of such a nucleophilic or electrophilic reactivefunctional group include, but are not limited to, a thiol, a primaryamine, a seleno, a hydrazide, an aldehyde, a carboxylic acid, a ketone,an aminooxy, a masked (protected) aldehyde, or a masked (protected) ketofunctional group. Examples of amino acid residues having a side chaincomprising a nucleophilic reactive functional group include, but are notlimited to, a lysine residue, a homolysine, an α,β-diaminopropionic acidresidue, an α,γ-diaminobutyric acid residue, an ornithine residue, acysteine, a homocysteine, a glutamic acid residue, an aspartic acidresidue, or a selenocysteine residue.

Amino acid residues are commonly categorized according to differentchemical and/or physical characteristics. The term “acidic amino acidresidue” refers to amino acid residues in D- or L-form having sidechains comprising acidic groups. Exemplary acidic residues includeaspartatic acid and glutamatic acid residues. The term “alkyl amino acidresidue” refers to amino acid residues in D- or L-form having C₁₋₆alkylside chains which may be linear, branched, or cyclized, including to theamino acid amine as in proline, wherein the C₁₋₆alkyl is substituted by0, 1, 2 or 3 substituents selected from C₁₋₄haloalkyl, halo, cyano,nitro, —C(═O)R^(b), —C(═O)OR^(a), —C(═O)NR^(a)R^(a),—C(═NR^(a))NR^(a)R^(a), —NR^(a)C(═NR^(a))NR^(a)R^(a), —OR^(a),—OC(═O)R^(b), —OC(═O)NR^(a)R^(a), —OC₂₋₆alkylNR^(a)R^(a),—OC₂₋₆alkylOR^(a), —SR^(a), —S(═O)R^(b), —S(═O)₂R^(b),—S(═O)₂NR^(a)R^(a), —NR^(a)R^(a), —N(R^(a))C(═O)R^(b),—N(R^(a))C(═O)OR^(b), —N(R^(a))C(═O)NR^(a)R^(a),—N(R^(a))C(═NR^(a))NR^(a)R^(a), —N(R^(a))S(═O)₂R^(b),—N(R^(a))S(═O)₂NR^(a)R^(a), —NR^(a)C₂₋₆alkylNR^(a)R^(a) and—NR^(a)C₂₋₆alkylOR^(a); wherein R^(a) is independently, at eachinstance, H or R^(b); and R^(b) is independently, at each instanceC₁₋₆alkyl substituted by 0, 1, 2 or 3 substituents selected from halo,C₁₋₄alk, C₁₋₃haloalk, —OC₁₋₄alk, —NH₂, —NHC₁₋₄alk, and—N(C₁₋₄alk)C₁₋₄alk; or any protonated form thereof, including alanine,valine, leucine, isoleucine, proline, serine, threonine, lysine,arginine, histidine, aspartate, glutamate, asparagine, glutamine,cysteine, methionine, hydroxyproline, but which residues do not containan aryl or aromatic group. The term “aromatic amino acid residue” refersto amino acid residues in D- or L-form having side chains comprisingaromatic groups. Exemplary aromatic residues include tryptophan,tyrosine, 3-(1-naphthyl)alanine, or phenylalanine residues. The term“basic amino acid residue” refers to amino acid residues in D- or L-formhaving side chains comprising basic groups. Exemplary basic amino acidresidues include histidine, lysine, homolysine, ornithine, arginine,N-methyl-arginine, co-aminoarginine, ω-methyl-arginine,1-methyl-histidine, 3-methyl-histidine, and homoarginine (hR) residues.The term “hydrophilic amino acid residue” refers to amino acid residuesin D- or L-form having side chains comprising polar groups. Exemplaryhydrophilic residues include cysteine, serine, threonine, histidine,lysine, asparagine, aspartate, glutamate, glutamine, and citrulline(Cit) residues. The terms “lipophilic amino acid residue” refers toamino acid residues in D- or L-form having sidechains comprisinguncharged, aliphatic or aromatic groups. Exemplary lipophilic sidechainsinclude phenylalanine, isoleucine, leucine, methionine, valine,tryptophan, and tyrosine. Alanine (A) is amphiphilic—it is capable ofacting as a hydrophilic or lipophilic residue. Alanine, therefore, isincluded within the definition of both “lipophilic residue” and“hydrophilic residue.” The term “nonfunctional amino acid residue”refers to amino acid residues in D- or L-form having side chains thatlack acidic, basic, or aromatic groups. Exemplary neutral amino acidresidues include methionine, glycine, alanine, valine, isoleucine,leucine, and norleucine (Nle) residues.

Additional useful embodiments of can result from conservativemodifications of the amino acid sequences of the polypeptides disclosedherein. Conservative modifications will produce half-life extendingmoiety-conjugated peptides having functional, physical, and chemicalcharacteristics similar to those of the conjugated (e.g.,PEG-conjugated) peptide from which such modifications are made. Suchconservatively modified forms of the conjugated polypeptides disclosedherein are also contemplated as being an embodiment of the presentinvention.

In contrast, substantial modifications in the functional and/or chemicalcharacteristics of peptides may be accomplished by selectingsubstitutions in the amino acid sequence that differ significantly intheir effect on maintaining (a) the structure of the molecular backbonein the region of the substitution, for example, as an α-helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the size of the molecule.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis” (see, forexample, MacLennan et al., Acta Physiol. Scand. Suppl., 643:55-67(1998); Sasaki et al., 1998, Adv. Biophys. 35:1-24 (1998), which discussalanine scanning mutagenesis).

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the peptidesequence, or to increase or decrease the affinity of the peptide orvehicle-conjugated peptide molecules described herein.

Naturally occurring residues may be divided into classes based on commonside chain properties:

1) hydrophobic: norleucine (Nor or Nle), Met, Ala, Val, Leu, Ile;2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;3) acidic: Asp, Glu;4) basic: His, Lys, Arg;5) residues that influence chain orientation: Gly, Pro; and6) aromatic: Tip, Tyr, Phe.

Conservative amino acid substitutions may involve exchange of a memberof one of these classes with another member of the same class.Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues may be introduced into regions of the toxin peptide analog.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art(see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,in certain embodiments, the substitution of amino acids whosehydropathic indices are within ±2 is included. In certain embodiments,those that are within ±1 are included, and in certain embodiments, thosewithin ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, asdisclosed herein. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those that are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One may also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine norleucine, alanine, or methionine for another, the substitutionof one polar (hydrophilic) amino acid residue for another such asbetween arginine and lysine, between glutamine and asparagine, betweenglycine and serine, the substitution of one basic amino acid residuesuch as lysine, arginine or histidine for another, or the substitutionof one acidic residue, such as aspartic acid or glutamic acid foranother. The phrase “conservative amino acid substitution” also includesthe use of a chemically derivatized residue in place of anon-derivatized residue, provided that such polypeptide displays therequisite bioactivity. Other exemplary amino acid substitutions that canbe useful in accordance with the present invention are set forth inTable 5 below.

TABLE 5 Some Useful Amino Acid Substitutions. Original ExemplaryResidues Substitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln AspGlu Cys Ser, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg IleLeu, Val, Met, Ala, Phe, Norleucine Leu Norleucine, Ile, Val, Met, Ala,Phe Lys Arg, 1,4-Diamino- butyric Acid, Gln, Asn Met Leu, Phe, Ile PheLeu, Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, PheTyr Trp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala, Norleucine

Ordinarily, amino acid sequence variants of the immunoglobulin will havean amino acid sequence having at least 60% amino acid sequence identitywith the original immunoglobulin or antibody amino acid sequences ofeither the heavy or the light chain variable region, or at least 65%, orat least 70%, or at least 75% or at least 80% identity, more preferablyat least 85% identity, even more preferably at least 90% identity, andmost preferably at least 95% identity, including for example, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, and 100%. Identity or homology with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical with the original sequence,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the immunoglobulin or antibody sequence shall be construed asaffecting sequence identity or homology.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intra-sequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean immunoglobulin with an N-terminal methionyl residue or theimmunoglobulin (including antibody or antibody fragment) fused to anepitope tag or a salvage receptor binding epitope. Other insertionalvariants of the immunoglobulin or antibody molecule include the fusionto a polypeptide which increases the serum half-life of theimmunoglobulin, e.g. at the N-terminus or C-terminus.

Examples of epitope tags include the flu HA tag polypeptide and itsantibody 12CA5 [Field et al., Mol. Cell. Biol. 8: 2159-2165 (1988)]; thec-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto[Evan et al., Mol. Cell. Biol. 5(12): 3610-3616 (1985)]; and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al.,Protein Engineering 3(6): 547-553 (1990)]. Other exemplary tags are apoly-histidine sequence, generally around six histidine residues, thatpermits isolation of a compound so labeled using nickel chelation. Otherlabels and tags, such as the FLAG® tag (Eastman Kodak, Rochester, N.Y.)are well known and routinely used in the art.

Some particular, non-limiting, embodiments of amino acid substitutionvariants of the inventive immunoglobulins, including antibodies andantibody fragments are exemplified below.

Any cysteine residue not involved in maintaining the proper conformationof the immunoglobulin also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking Conversely, cysteine bond(s) may be added to theimmunoglobulin to improve its stability (particularly where theimmunoglobulin is an antibody fragment such as an Fv fragment).

In certain instances, immunoglobulin variants are prepared with theintent to modify those amino acid residues which are directly involvedin epitope binding in a starting sequence. In other embodiments,modification of residues which are not directly involved in epitopebinding or residues not involved in epitope binding in any way, isdesirable, for purposes discussed herein. Mutagenesis within any of theCDR regions and/or framework regions is contemplated.

In order to determine which antigen binding protein amino acid residuesare important for epitope recognition and binding, alanine scanningmutagenesis can be performed to produce substitution variants. See, forexample, Cunningham et al., Science, 244:1081-1085 (1989), thedisclosure of which is incorporated herein by reference in its entirety.In this method, individual amino acid residues are replacedone-at-a-time with an alanine residue and the resulting antibody isscreened for its ability to bind its specific epitope relative to theunmodified polypeptide. Modified antigen binding proteins with reducedbinding capacity are sequenced to determine which residue was changed,indicating its significance in binding or biological properties.

Substitution variants of antigen binding proteins can be prepared byaffinity maturation wherein random amino acid changes are introducedinto the parent polypeptide sequence. See, for example, Ouwehand et al.,Vox Sang 74 (Suppl 2):223-232, 1998; Rader et al., Proc. Natl. Acad.Sci. USA 95:8910-8915, 1998; Dall′Acqua et al., Curr. Opin. Struct.Biol. 8:443-450, 1998, the disclosures of which are incorporated hereinby reference in their entireties. Affinity maturation involves preparingand screening the antigen binding proteins, or variants thereof andselecting from the resulting variants those that have modifiedbiological properties, such as increased binding affinity relative tothe parent antigen binding protein. A convenient way for generatingsubstitutional variants is affinity maturation using phage display.Briefly, several hypervariable region sites are mutated to generate allpossible amino substitutions at each site. The variants thus generatedare expressed in a monovalent fashion on the surface of filamentousphage particles as fusions to the gene III product of M13 packagedwithin each particle. The phage-displayed variants are then screened fortheir biological activity (e.g., binding affinity). See e.g., WO92/01047, WO 93/112366, WO 95/15388 and WO 93/19172.

Current antibody affinity maturation methods belong to two mutagenesiscategories: stochastic and nonstochastic. Error prone PCR, mutatorbacterial strains (Low et al., J. Mol. Biol. 260, 359-68, 1996), andsaturation mutagenesis (Nishimiya et al., J. Biol. Chem. 275:12813-20,2000; Chowdhury, P. S. Methods Mol. Biol. 178, 269-85, 2002) are typicalexamples of stochastic mutagenesis methods (Rajpal et al., Proc NatlAcad Sci USA. 102:8466-71, 2005). Nonstochastic techniques often usealanine-scanning or site-directed mutagenesis to generate limitedcollections of specific muteins. Some methods are described in furtherdetail below.

Affinity Maturation Via Panning Methods—

Affinity maturation of recombinant antibodies is commonly performedthrough several rounds of panning of candidate antibodies in thepresence of decreasing amounts of antigen. Decreasing the amount ofantigen per round selects the antibodies with the highest affinity tothe antigen thereby yielding antibodies of high affinity from a largepool of starting material. Affinity maturation via panning is well knownin the art and is described, for example, in Huls et al. (Cancer ImmunolImmunother. 50:163-71, 2001). Methods of affinity maturation using phagedisplay technologies are described elsewhere herein and known in the art(see e.g., Daugherty et al., Proc Natl Acad Sci USA. 97:2029-34, 2000).

Look-through mutagenesis—Look-through mutagenesis (LTM) (Rajpal et al.,Proc Natl Acad Sci USA. 102:8466-71, 2005) provides a method for rapidlymapping the antibody-binding site. For LTM, nine amino acids,representative of the major side-chain chemistries provided by the 20natural amino acids, are selected to dissect the functional side-chaincontributions to binding at every position in all six CDRs of anantibody. LTM generates a positional series of single mutations within aCDR where each “wild type” residue is systematically substituted by oneof nine selected amino acids. Mutated CDRs are combined to generatecombinatorial single-chain variable fragment (scFv) libraries ofincreasing complexity and size without becoming prohibitive to thequantitative display of all muteins. After positive selection, cloneswith improved binding are sequenced, and beneficial mutations aremapped.

Error-prone PCR—Error-prone PCR involves the randomization of nucleicacids between different selection rounds. The randomization occurs at alow rate by the intrinsic error rate of the polymerase used but can beenhanced by error-prone PCR (Zaccolo et al., J. Mol. Biol. 285:775-783,1999) using a polymerase having a high intrinsic error rate duringtranscription (Hawkins et al., J Mol Biol. 226:889-96, 1992). After themutation cycles, clones with improved affinity for the antigen areselected using routine methods in the art.

Techniques utilizing gene shuffling and directed evolution may also beused to prepare and screen antigen binding proteins, or variantsthereof, for desired activity. For example, Jermutus et al., Proc NatlAcad Sci USA., 98(1):75-80 (2001) showed that tailored in vitroselection strategies based on ribosome display were combined with invitro diversification by DNA shuffling to evolve either the off-rate orthermodynamic stability of scFvs; Fermer et al., Tumour Biol. 2004January-April; 25(1-2):7-13 reported that use of phage display incombination with DNA shuffling raised affinity by almost three orders ofmagnitude. Dougherty et al., Proc Natl Acad Sci USA. 2000 Feb. 29;97(5):2029-2034 reported that (i) functional clones occur at anunexpectedly high frequency in hypermutated libraries, (ii)gain-of-function mutants are well represented in such libraries, and(iii) the majority of the scFv mutations leading to higher affinitycorrespond to residues distant from the binding site.

Alternatively, or in addition, it may be beneficial to analyze a crystalstructure of the antigen-antibody complex to identify contact pointsbetween the antibody and antigen, or to use computer software to modelsuch contact points. Such contact residues and neighboring residues arecandidates for substitution according to the techniques elaboratedherein. Once such variants are generated, they are subjected toscreening as described herein and antibodies with superior properties inone or more relevant assays may be selected for further development.

Immunoglobulins with Modified Carbohydrate

Immunoglobulin variants can also be produced that have a modifiedglycosylation pattern relative to the parent polypeptide, for example,adding or deleting one or more of the carbohydrate moieties bound to theimmunoglobulin, and/or adding or deleting one or more glycosylationsites in the immunoglobulin.

Glycosylation of polypeptides, including antibodies is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. The presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. Thus, N-linkedglycosylation sites may be added to an immunoglobulin by altering theamino acid sequence such that it contains one or more of thesetripeptide sequences. O-linked glycosylation refers to the attachment ofone of the sugars N-aceylgalactosamine, galactose, or xylose to ahydroxyamino acid, most commonly serine or threonine, although5-hydroxyproline or 5-hydroxylysine may also be used. O-linkedglycosylation sites may be added to an immunoglobulin by inserting orsubstituting one or more serine or threonine residues to the sequence ofthe original immunoglobulin or antibody.

Altered Effector Function

Cysteine residue(s) may be removed or introduced in the Fc region of anantibody or Fc-containing polypeptide, thereby eliminating or increasinginterchain disulfide bond formation in this region. A homodimericimmunoglobulin thus generated may have improved internalizationcapability and/or increased complement-mediated cell killing andantibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J.Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922(1992). Homodimeric immunoglobulins or antibodies may also be preparedusing heterobifunctional cross-linkers as described in Wolff et al.,Cancer Research 53: 2560-2565 (1993). Alternatively, an immunoglobulincan be engineered which has dual Fc regions and may thereby haveenhanced complement lysis and ADCC capabilities. See Stevenson et al.,Anti-CancerDrug Design 3: 219-230 (1989).

It is also contemplated that one or more of the N-terminal 20 amino acidresidues (e.g., a signal sequence) of the heavy or light chain areremoved.

Modifications to increase serum half-life also may desirable, forexample, by incorporation of or addition of a salvage receptor bindingepitope (e.g., by mutation of the appropriate region or by incorporatingthe epitope into a peptide tag that is then fused to the immunoglobulinat either end or in the middle, e.g., by DNA or peptide synthesis) (see,e.g., WO96/32478) or adding molecules such as PEG or other water solublepolymers, including polysaccharide polymers.

The salvage receptor binding epitope preferably constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the immunoglobulinor fragment. Even more preferably, three or more residues from one ortwo loops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or VH region, or more than one suchregion, of the immunoglobulin or antibody. Alternatively, the epitope istaken from the CH2 domain of the Fc region and transferred to the C_(L)region or V_(L) region, or both, of the immunoglobulin fragment. Seealso International applications WO 97/34631 and WO 96/32478 whichdescribe Fc variants and their interaction with the salvage receptor.

Other sites and amino acid residue(s) of the constant region have beenidentified that are responsible for complement dependent cytotoxicity(CDC), such as the Clq binding site, and/or the antibody-dependentcellular cytotoxicity (ADCC) [see, e.g., Molec. Immunol. 29 (5): 633-9(1992); Shields et al., J. Biol. Chem., 276(9):6591-6604 (2001); Lazaret al., Proc. Nat'l. Acad. Sci. 103(11): 4005 (2006) which describe theeffect of mutations at specific positions, each of which is incorporatedby reference herein in its entirety]. Mutation of residues within Fcreceptor binding sites can result in altered (i.e. increased ordecreased) effector function, such as altered affinity for Fc receptors,altered ADCC or CDC activity, or altered half-life. As described above,potential mutations include insertion, deletion or substitution of oneor more residues, including substitution with alanine, a conservativesubstitution, a non-conservative substitution, or replacement with acorresponding amino acid residue at the same position from a differentsubclass (e.g. replacing an IgG1 residue with a corresponding IgG2residue at that position).

The invention also encompasses production of immunoglobulin molecules,including antibodies and antibody fragments, with altered carbohydratestructure resulting in altered effector activity, including antibodymolecules with absent or reduced fucosylation that exhibit improved ADCCactivity. A variety of ways are known in the art to accomplish this. Forexample, ADCC effector activity is mediated by binding of the antibodymolecule to the FcγRIII receptor, which has been shown to be dependenton the carbohydrate structure of the N-linked glycosylation at theAsn-297 of the CH2 domain. Non-fucosylated antibodies bind this receptorwith increased affinity and trigger FcγRIII-mediated effector functionsmore efficiently than native, fucosylated antibodies. For example,recombinant production of non-fucosylated antibody in CHO cells in whichthe alpha-1,6-fucosyl transferase enzyme has been knocked out results inantibody with 100-fold increased ADCC activity (Yamane-Ohnuki et al.,Biotechnol Bioeng. 2004 Sep. 5; 87(5):614-22). Similar effects can beaccomplished through decreasing the activity of this or other enzymes inthe fucosylation pathway, e.g., through siRNA or antisense RNAtreatment, engineering cell lines to knockout the enzyme(s), orculturing with selective glycosylation inhibitors (Rothman et al., MolImmunol. 1989 December; 26(12):1113-23). Some host cell strains, e.g.Lec13 or rat hybridoma YB2/0 cell line naturally produce antibodies withlower fucosylation levels. Shields et al., J Biol Chem. 2002 Jul. 26;277(30):26733-40; Shinkawa et al., J Biol Chem. 2003 Jan. 31;278(5):3466-73. An increase in the level of bisected carbohydrate, e.g.through recombinantly producing antibody in cells that overexpressGnTIII enzyme, has also been determined to increase ADCC activity.

Umana et al., Nat Biotechnol. 1999 February; 17(2):176-80. It has beenpredicted that the absence of only one of the two fucose residues may besufficient to increase ADCC activity. (Ferrara et al., J Biol Chem. 2005Dec. 5).

Other Covalent Modifications of Immunoglobulins

Other particular covalent modifications of the immunoglobulin, are alsoincluded within the scope of this invention. They may be made bychemical synthesis or by enzymatic or chemical cleavage of theimmunoglobulin or antibody, if applicable. Other types of covalentmodifications can be introduced by reacting targeted amino acid residueswith an organic derivatizing agent that is capable of reacting withselected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,.alpha.-bromo-13-(5-imidozoyl) propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing .alpha.-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pk_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N.dbd.C.dbd.N—R′), where R and R′ aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the .alpha.-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the immunoglobulin (e.g., antibodyor antibody fragment). These procedures are advantageous in that they donot require production of the immunoglobulin in a host cell that hasglycosylation capabilities for N- or O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO87/05330 published 11 Sep.1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306(1981).

Removal of any carbohydrate moieties present on the immunoglobulin maybe accomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the immunoglobulin to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theimmunoglobulin intact. Chemical deglycosylation is described byHakimuddin, et al. Arch. Biochem. Biophys. 259: 52 (1987) and by Edge etal. Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydratemoieties on an immunoglobulin can be achieved by the use of a variety ofendo- and exo-glycosidases as described by Thotakura et al. Meth.Enzymol. 138: 350 (1987).

Another type of covalent modification of the immunoglobulins of theinvention (including antibodies and antibody fragments) compriseslinking the immunoglobulin to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol, polypropylene glycol,polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol, polyoxyalkylenes, or polysaccharidepolymers such as dextran. Such methods are known in the art, see, e.g.U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192,4,179,337, 4,766,106, 4,179,337, 4,495,285, 4,609,546 or EP 315 456.

Isolated Nucleic Acids

Another aspect of the present invention is an isolated nucleic acid thatencodes an immunoglobulin of the invention, such as, but not limited to,an isolated nucleic acid that encodes an antibody or antibody fragmentof the invention. Such nucleic acids are made by recombinant techniquesknown in the art and/or disclosed herein.

In other embodiments the isolated nucleic acid encodes an immunoglobulincomprising an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region, wherein:

(a) the heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:323 and the light chain variable region comprises the aminoacid sequence of SEQ ID NO:188 or SEQ ID NO:190; or

(b) the heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:321 and the light chain variable region comprises the aminoacid sequence of SEQ ID NO:188 or SEQ ID NO:190; or

(c) the heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:325 and the light chain variable region comprises the aminoacid sequence of SEQ ID NO:182, SEQ ID NO:188, or SEQ ID NO:190.

And in some embodiments the isolated nucleic acid encodes animmunoglobulin comprising comprising an immunoglobulin heavy chainvariable region and an immunoglobulin light chain variable region,wherein:

(a) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:196 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:335, SEQ ID NO:349, SEQ ID NO:351, SEQ IDNO:353, SEQ ID NO:355, or SEQ ID NO:359; or

(b) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:204 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:349 or SEQ ID NO:355; or

(c) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:202 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:349; or

(d) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:192 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:357, SEQ ID NO:359, or SEQ ID NO:369; or

(e) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:194 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:335, SEQ ID NO:349, or SEQ ID NO:351.

In other examples, the isolated nucleic acid encodes an immunoglobulincomprising an immunoglobulin heavy chain and an immunoglobulin lightchain wherein:

(a) the heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:323; and the light chain variable region comprises the aminoacid sequence of SEQ ID NO:188; or

(b) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:196; and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:353; or

(c) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:202; and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:349; or

(d) the heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:325; and the light chain variable region comprises the aminoacid sequence of SEQ ID NO:190.

Or in some embodiments, the isolated nucleic acid encodes animmunoglobulin comprising:

an immunoglobulin heavy chain comprising the amino acid sequence of SEQID NO:113, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminalor C-terminal, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:110, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminal or C-terminal, or both; or

an immunoglobulin heavy chain comprising the amino acid sequence of SEQID NO:125, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminalor C-terminal, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:122, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminal or C-terminal, or both; or

an immunoglobulin heavy chain comprising the amino acid sequence of SEQID NO:101, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminalor C-terminal, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:98, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminal or C-terminal, or both; or

an immunoglobulin heavy chain comprising the amino acid sequence of SEQID NO:119, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminalor C-terminal, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:116, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminal or C-terminal, or both.

The present invention is also directed to vectors, including expressionvectors that comprise any of the inventive isolated nucleic acids. Anisolated host cell that comprises the expression vector is alsoencompassed by the present invention, which is made by molecularbiological techniques known in the art and/or disclosed herein.

The invention is also directed to a method involving:

culturing the host cell in a culture medium under conditions permittingexpression of the immunoglobulin encoded by the expression vector; and

recovering the immunoglobulin from the culture medium. Recovering theimmunoglobulin is accomplished by known methods of antbody purification,such as but not limited to, antibody purification techniques disclosedin Example 1 and elsewhere herein.

Gene Therapy

Delivery of a therapeutic immunoglobulin to appropriate cells can beeffected via gene therapy ex vivo, in situ, or in vivo by use of anysuitable approach known in the art. For example, for in vivo therapy, anucleic acid encoding the desired immunoglobulin or antibody, eitheralone or in conjunction with a vector, liposome, or precipitate may beinjected directly into the subject, and in some embodiments, may beinjected at the site where the expression of the immunoglobulin compoundis desired. For ex vivo treatment, the subject's cells are removed, thenucleic acid is introduced into these cells, and the modified cells arereturned to the subject either directly or, for example, encapsulatedwithin porous membranes which are implanted into the patient. See, e.g.U.S. Pat. Nos. 4,892,538 and 5,283,187.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, chemical treatments,DEAE-dextran, and calcium phosphate precipitation. Other in vivo nucleicacid transfer techniques include transfection with viral vectors (suchas adenovirus, Herpes simplex I virus, adeno-associated virus orretrovirus) and lipid-based systems. The nucleic acid and transfectionagent are optionally associated with a microparticle. Exemplarytransfection agents include calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, quaternaryammonium amphiphile DOTMA ((dioleoyloxypropyl) trimethylammoniumbromide, commercialized as Lipofectin by GIBCO-BRL))(Felgner et al,(1987) Proc. Natl. Acad. Sci. USA 84, 7413-7417; Malone et al. (1989)Proc. Natl Acad. Sci. USA 86 6077-6081); lipophilic glutamate diesterswith pendent trimethylammonium heads (Ito et al. (1990) Biochem.Biophys. Acta 1023, 124-132); the metabolizable parent lipids such asthe cationic lipid dioctadecylamido glycylspermine (DOGS, Transfectam,Promega) and dipalmitoylphosphatidyl ethanolamylspermine (DPPES)(J. P.Behr (1986) Tetrahedron Lett. 27, 5861-5864; J. P. Behr et al. (1989)Proc. Natl. Acad. Sci. USA 86, 6982-6986); metabolizable quaternaryammonium salts (DOTB,N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate(DOTAP)(Boehringer Mannheim), polyethyleneimine (PEI), dioleoyl esters,ChoTB, ChoSC, DOSC)(Leventis et al. (1990) Biochim. Inter. 22, 235-241);3beta[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol),dioleoylphosphatidyl ethanolamine(DOPE)/3beta[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterolDC-Cholin one to one mixtures (Gao et al., (1991) Biochim. Biophys. Acta 1065,8-14), spermine, spermidine, lipopolyamines (Behr et al., BioconjugateChem, 1994, 5: 382-389), lipophilic polylysines (LPLL) (Zhou et al.,(1991) Biochim. Biophys. Acta 939, 8-18),[[(1,1,3,3-tetramethylbutyl)cre-soxy]ethoxy]ethyl]dimethylbenzylammoniumhydroxide (DEBDA hydroxide) with excess phosphatidylcholine/cholesterol(Ballas et al., (1988) Biochim. Biophys. Acta 939, 8-18),cetyltrimethylammonium bromide (CTAB)/DOPE mixtures (Pinnaduwage et al,(1989) Biochim. Biophys. Acta 985, 33-37), lipophilic diester ofglutamic acid (TMAG) with DOPE, CTAB, DEBDA, didodecylammonium bromide(DDAB), and stearylamine in admixture with phosphatidylethanolamine(Rose et al., (1991) Biotechnique 10, 520-525), DDAB/DOPE (TransfectACE,GIBCO BRL), and oligogalactose bearing lipids. Exemplary transfectionenhancer agents that increase the efficiency of transfer include, forexample, DEAE-dextran, polybrene, lysosome-disruptive peptide (Ohmori NI et al, Biochem Biophys Res Commun Jun. 27, 1997; 235(3):726-9),chondroitan-based proteoglycans, sulfated proteoglycans,polyethylenimine, polylysine (Pollard H et al. J Biol Chem, 1998 273(13):7507-11), integrin-binding peptide CYGGRGDTP (SEQ ID NO:235),linear dextran nonasaccharide, glycerol, cholesteryl groups tethered atthe 3′-terminal internucleoside link of an oligonucleotide (Letsinger,R. L. 1989 Proc Natl Acad Sci USA 86: (17):6553-6), lysophosphatide,lysophosphatidylcholine, lysophosphatidylethanolamine, and 1-oleoyllysophosphatidylcholine.

In some situations it may be desirable to deliver the nucleic acid withan agent that directs the nucleic acid-containing vector to targetcells. Such “targeting” molecules include antigen binding proteinsspecific for a cell-surface membrane protein on the target cell, or aligand for a receptor on the target cell. Where liposomes are employed,proteins which bind to a cell-surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake.Examples of such proteins include capsid proteins and fragments thereoftropic for a particular cell type, antigen binding proteins for proteinswhich undergo internalization in cycling, and proteins that targetintracellular localization and enhance intracellular half-life. In otherembodiments, receptor-mediated endocytosis can be used. Such methods aredescribed, for example, in Wu et al., 1987 or Wagner et al., 1990. Forreview of the currently known gene marking and gene therapy protocols,see Anderson 1992. See also WO 93/25673 and the references citedtherein. For additional reviews of gene therapy technology, seeFriedmann, Science, 244: 1275-1281 (1989); Anderson, Nature, supplementto vol. 392, no 6679, pp. 25-30 (1998); Verma, Scientific American:68-84 (1990); and Miller, Nature, 357: 455460 (1992).

Administration and Preparation of Pharmaceutical Formulations

The immunoglobulins or antibodies used in the practice of a method ofthe invention may be formulated into pharmaceutical compositions andmedicaments comprising a carrier suitable for the desired deliverymethod. Suitable carriers include any material which, when combined withthe immunoglobulin or antibody, and is nonreactive with the subject'simmune systems. Examples include, but are not limited to, any of anumber of standard pharmaceutical carriers such as sterile phosphatebuffered saline solutions, bacteriostatic water, and the like. A varietyof aqueous carriers may be used, e.g., water, buffered water, 0.4%saline, 0.3% glycine and the like, and may include other proteins forenhanced stability, such as albumin, lipoprotein, globulin, etc.,subjected to mild chemical modifications or the like.

Exemplary immunoglobulin concentrations in the formulation may rangefrom about 0.1 mg/ml to about 180 mg/ml or from about 0.1 mg/mL to about50 mg/mL, or from about 0.5 mg/mL to about 25 mg/mL, or alternativelyfrom about 2 mg/mL to about 10 mg/mL. An aqueous formulation of theimmunoglobulin may be prepared in a pH-buffered solution, for example,at pH ranging from about 4.5 to about 6.5, or from about 4.8 to about5.5, or alternatively about 5.0. Examples of buffers that are suitablefor a pH within this range include acetate (e.g. sodium acetate),succinate (such as sodium succinate), gluconate, histidine, citrate andother organic acid buffers. The buffer concentration can be from about 1mM to about 200 mM, or from about 10 mM to about 60 mM, depending, forexample, on the buffer and the desired isotonicity of the formulation.

A tonicity agent, which may also stabilize the immunoglobulin, may beincluded in the formulation. Exemplary tonicity agents include polyols,such as mannitol, sucrose or trehalose. Preferably the aqueousformulation is isotonic, although hypertonic or hypotonic solutions maybe suitable. Exemplary concentrations of the polyol in the formulationmay range from about 1% to about 15% w/v.

A surfactant may also be added to the immunoglobulin formulation toreduce aggregation of the formulated immunoglobulin and/or minimize theformation of particulates in the formulation and/or reduce adsorption.Exemplary surfactants include nonionic surfactants such as polysorbates(e.g. polysorbate 20, or polysorbate 80) or poloxamers (e.g. poloxamer188). Exemplary concentrations of surfactant may range from about 0.001%to about 0.5%, or from about 0.005% to about 0.2%, or alternatively fromabout 0.004% to about 0.01% w/v.

In one embodiment, the formulation contains the above-identified agents(i.e. immunoglobulin, buffer, polyol and surfactant) and is essentiallyfree of one or more preservatives, such as benzyl alcohol, phenol,m-cresol, chlorobutanol and benzethonium Cl. In another embodiment, apreservative may be included in the formulation, e.g., at concentrationsranging from about 0.1% to about 2%, or alternatively from about 0.5% toabout 1%. One or more other pharmaceutically acceptable carriers,excipients or stabilizers such as those described in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may beincluded in the formulation provided that they do not adversely affectthe desired characteristics of the formulation. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed and include; additional buffering agents;co-solvents; antoxidants including ascorbic acid and methionine;chelating agents such as EDTA; metal complexes (e.g. Zn-proteincomplexes); biodegradable polymers such as polyesters; and/orsalt-forming counterions such as sodium.

Therapeutic formulations of the immunoglobulin are prepared for storageby mixing the immunoglobulin having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose,maltose, or dextrins; chelating agents such as EDTA; sugars such assucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions suchas sodium; metal complexes (e.g., Zn-protein complexes); and/ornon-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol(PEG).

In one embodiment, a suitable formulation of the claimed inventioncontains an isotonic buffer such as a phosphate, acetate, or Tris bufferin combination with a tonicity agent such as a polyol, Sorbitol, sucroseor sodium chloride which tonicifies and stabilizes. One example of sucha tonicity agent is 5% Sorbitol or sucrose. In addition, the formulationcould optionally include a surfactant such as to prevent aggregation andfor stabilization at 0.01 to 0.02% wt/vol. The pH of the formulation mayrange from 4.5-6.5 or 4.5 to 5.5. Other exemplary descriptions ofpharmaceutical formulations for antibodies may be found in US2003/0113316 and U.S. Pat. No. 6,171,586, each incorporated herein byreference in its entirety.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Suspensions and crystal forms of immunoglobulins are also contemplated.Methods to make suspensions and crystal forms are known to one of skillin the art.

The formulations to be used for in vivo administration must be sterile.The compositions of the invention may be sterilized by conventional,well known sterilization techniques. For example, sterilization isreadily accomplished by filtration through sterile filtration membranes.The resulting solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile solution prior to administration.

The process of freeze-drying is often employed to stabilize polypeptidesfor long-term storage, particularly when the polypeptide is relativelyunstable in liquid compositions. A lyophilization cycle is usuallycomposed of three steps: freezing, primary drying, and secondary drying;Williams and Polli, Journal of Parenteral Science and Technology, Volume38, Number 2, pages 48-59 (1984). In the freezing step, the solution iscooled until it is adequately frozen. Bulk water in the solution formsice at this stage. The ice sublimes in the primary drying stage, whichis conducted by reducing chamber pressure below the vapor pressure ofthe ice, using a vacuum. Finally, sorbed or bound water is removed atthe secondary drying stage under reduced chamber pressure and anelevated shelf temperature. The process produces a material known as alyophilized cake. Thereafter the cake can be reconstituted prior to use.

The standard reconstitution practice for lyophilized material is to addback a volume of pure water (typically equivalent to the volume removedduring lyophilization), although dilute solutions of antibacterialagents are sometimes used in the production of pharmaceuticals forparenteral administration; Chen, Drug Development and IndustrialPharmacy, Volume 18, Numbers 11 and 12, pages 1311-1354 (1992).

Excipients have been noted in some cases to act as stabilizers forfreeze-dried products; Carpenter et al., Developments in BiologicalStandardization, Volume 74, pages 225-239 (1991). For example, knownexcipients include polyols (including mannitol, sorbitol and glycerol);sugars (including glucose and sucrose); and amino acids (includingalanine, glycine and glutamic acid).

In addition, polyols and sugars are also often used to protectpolypeptides from freezing and drying-induced damage and to enhance thestability during storage in the dried state. In general, sugars, inparticular disaccharides, are effective in both the freeze-dryingprocess and during storage. Other classes of molecules, including mono-and di-saccharides and polymers such as PVP, have also been reported asstabilizers of lyophilized products.

For injection, the pharmaceutical formulation and/or medicament may be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze dried, rotary dried or spray dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin, which matrices arein the form of shaped articles, e.g., films, or microcapsule. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LupronDepot™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulatedpolypeptides remain in the body for a long time, they may denature oraggregate as a result of exposure to moisture at 37° C., resulting in aloss of biological activity and possible changes in immunogenicity.Rational strategies can be devised for stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

The formulations of the invention may be designed to be short-acting,fast-releasing, long-acting, or sustained-releasing as described herein.Thus, the pharmaceutical formulations may also be formulated forcontrolled release or for slow release.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention.

The immunoglobulin is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intravenous, intraarterial,intraperitoneal, intramuscular, intradermal or subcutaneousadministration. In addition, the immunoglobulin is suitably administeredby pulse infusion, particularly with declining doses of theimmunoglobulin or antibody. Preferably the dosing is given byinjections, most preferably intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.Other administration methods are contemplated, including topical,particularly transdermal, transmucosal, rectal, oral or localadministration e.g. through a catheter placed close to the desired site.Most preferably, the immunoglobulin of the invention is administeredintravenously in a physiological solution at a dose ranging between 0.01mg/kg to 100 mg/kg at a frequency ranging from daily to weekly tomonthly (e.g. every day, every other day, every third day, or 2, 3, 4,5, or 6 times per week), preferably a dose ranging from 0.1 to 45 mg/kg,0.1 to 15 mg/kg or 0.1 to 10 mg/kg at a frequency of 2 or 3 times perweek, or up to 45 mg/kg once a month.

Embodiments or aspects of the invention can include but are not limitedto the following:

Aspect 1. An isolated immunoglobulin, comprising an immunoglobulin heavychain variable region and an immunoglobulin light chain variable region,wherein:

(a) the heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:323 and the light chain variable region comprises the aminoacid sequence of SEQ ID NO:188 or SEQ ID NO:190; or

(b) the heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:321 and the light chain variable region comprises the aminoacid sequence of SEQ ID NO:188 or SEQ ID NO:190; or

(c) the heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:325 and the light chain variable region comprises the aminoacid sequence of SEQ ID NO:182, SEQ ID NO:188, or SEQ ID NO:190.

Aspect 2. An isolated immunoglobulin, comprising an immunoglobulin heavychain variable region and an immunoglobulin light chain variable region,wherein:

(a) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:196 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:335, SEQ ID NO:349, SEQ ID NO:351, SEQ IDNO:353, SEQ ID NO:355, or SEQ ID NO:359; or

(b) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:204 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:349 or SEQ ID NO:355; or

(c) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:202 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:349; or

(d) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:192 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:357, SEQ ID NO:359, or SEQ ID NO:369; or

(e) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:194 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:335, SEQ ID NO:349, or SEQ ID NO:351.

Aspect 3. The isolated immunoglobulin of Aspect 1, wherein the heavychain variable region comprises the amino acid sequence of SEQ IDNO:323; and the light chain variable region comprises the amino acidsequence of SEQ ID NO:188.

Aspect 4. The isolated immunoglobulin of Aspect 2, wherein the lightchain variable region comprises the amino acid sequence of SEQ IDNO:196; and the heavy chain variable region comprises the amino acidsequence of SEQ ID NO:353.

Aspect 5. The isolated immunoglobulin of Aspect 2, wherein the lightchain variable region comprises the amino acid sequence of SEQ IDNO:202; and the heavy chain variable region comprises the amino acidsequence of SEQ ID NO:349.

Aspect 6. The isolated immunoglobulin of Aspect 1, wherein the heavychain variable region comprises the amino acid sequence of SEQ IDNO:325; and the light chain variable region comprises the amino acidsequence of SEQ ID NO:190.

Aspect 7. The isolated immunoglobulin of Aspect 1 or Aspect 2, whereinthe isolated immunoglobulin comprises an antibody or antibody fragment.

Aspect 8. The isolated immunoglobulin of any of Aspects 1-7, comprisingan IgG1, IgG2, IgG3 or IgG4.

Aspect 9. The isolated immunoglobulin of any of Aspects 1-8, comprisinga monoclonal antibody.

Aspect 10. The isolated immunoglobulin of any of Aspects 1-9, comprisinga human antibody.

Aspect 11. The isolated immunoglobulin of Aspect 10, comprising:

(a) an immunoglobulin heavy chain comprising the amino acid sequence ofSEQ ID NO:113, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminalor C-terminal, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:110, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminal or C-terminal, or both; or

(b) an immunoglobulin heavy chain comprising the amino acid sequence ofSEQ ID NO:125, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminalor C-terminal, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:122, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminal or C-terminal, or both; or

(c) an immunoglobulin heavy chain comprising the amino acid sequence ofSEQ ID NO:101, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminalor C-terminal, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:98, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminal or C-terminal, or both; or

(d) an immunoglobulin heavy chain comprising the amino acid sequence ofSEQ ID NO:119, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminalor C-terminal, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:116, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminal or C-terminal, or both.

Aspect 12. The isolated immunoglobulin of any of Aspects 1-11, furthercomprising one to twenty-four pharmacologically active chemical moietiesconjugated thereto.

Aspect 13. The isolated immunoglobulin of any of Aspects 1-12, whereinthe pharmacologically active chemical moiety is a pharmacologicallyactive polypeptide.

Aspect 14. The isolated immunoglobulin of any of Aspects 1-13, whereinthe immunoglobulin is recombinantly produced.

Aspect 15. The isolated immunoglobulin of Aspect 14, wherein theimmunoglobulin comprises at least one immunoglobulin heavy chain and atleast one immunoglobulin light chain, and wherein the pharmacologicallyactive polypeptide is inserted in the primary amino acid sequence of theof the immunoglobulin heavy chain within an internal loop of the Fcdomain of the immunoglobulin heavy chain.

Aspect 16. The isolated immunoglobulin of Aspect 13 or 14, wherein theimmunoglobulin comprises at least one immunoglobulin heavy chain and atleast one immunoglobulin light chain, and wherein the pharmacologicallyactive polypeptide is conjugated at the N-terminal or C-terminal of theimmunoglobulin heavy chain.

Aspect 17. The isolated immunoglobulin of Aspect 13 or 14, wherein theimmunoglobulin comprises at least one immunoglobulin heavy chain and atleast one immunoglobulin light chain, and wherein the pharmacologicallyactive polypeptide is conjugated at the N-terminal or C-terminal of theimmunoglobulin light chain.

Aspect 18. The isolated immunoglobulin of Aspect 13 or 14, wherein thepharmacologically active polypeptide is a toxin peptide, an IL-6 bindingpeptide, a CGRP peptide antagonist, a bradykinin B1 receptor peptideantagonist, a PTH agonist peptide, a PTH antagonist peptide, an ang-1binding peptide, an ang-2 binding peptide, a myostatin binding peptide,an EPO-mimetic peptide, a FGF21 peptide, a TPO-mimetic peptide, a NGFbinding peptide, a BAFF antagonist peptide, a GLP-1 or peptide mimeticthereof, or a GLP-2 or peptide mimetic thereof.

Aspect 19. The isolated immunoglobulin of Aspect 18, wherein the toxinpeptide is ShK or a ShK peptide analog.

Aspect 20. A pharmaceutical composition comprising the immunoglobulin ofany of Aspects 1-19; and a pharmaceutically acceptable diluent,excipient or carrier.

Aspect 21. An isolated nucleic acid that encodes the immunoglobulin ofany of Aspects 1-11.

Aspect 22. An isolated nucleic acid that encodes the immunoglobulin ofAspect 3.

Aspect 23. An isolated nucleic acid that encodes the immunoglobulin ofAspect 4.

Aspect 24. An isolated nucleic acid that encodes the immunoglobulin ofAspect 5.

Aspect 25. An isolated nucleic acid that encodes the immunoglobulin ofAspect 6.

Aspect 26. An isolated nucleic acid that encodes the immunoglobulin ofAspect 11.

Aspect 27. An isolated nucleic acid that encodes the immunoglobulin ofany of Aspects 13-19.

Aspect 28. A vector comprising the isolated nucleic acid of Aspect 21.

Aspect 29. A vector comprising the isolated nucleic acid of any ofAspects 22-26.

Aspect 30. A vector comprising the isolated nucleic acid of Aspect 27.

Aspect 31. The vector of Aspect 28, comprising an expression vector.

Aspect 32. The vector of Aspect 29, comprising an expression vector.

Aspect 33. The vector of Aspect 30, comprising an expression vector.

Aspect 34. An isolated host cell, comprising the expression vector ofany of Aspects 31-33.

Aspect 35. A method, comprising:

(a) culturing the host cell of claim 34 in a culture medium underconditions permitting expression of the immunoglobulin encoded by theexpression vector; and

(b) recovering the immunoglobulin from the culture medium.

Aspect 36. The immunoglobulin of Aspect 1, wherein the immunoglobulin at30 micromolar concentration does not significantly bind soluble humanIL-17R (SEQ ID NO:89) at 30 nanomolar concentration in an aqueoussolution incubated under physiological conditions, as measured by asurface plasmon resonance binding assay.

Aspect 37. The immunoglobulin of Aspect 2, wherein the immunoglobulin at10 micromolar concentration does not significantly bind soluble humanTR2 (SEQ ID NO:82) at 10 nanomolar concentration in an aqueous solutionincubated under physiological conditions, as measured by a surfaceplasmon resonance binding assay.

The invention is illustrated by the following further examples, whichare not intended to be limiting in any way.

EXAMPLES Example 1 Generation of Antibodies to Human IL-17R andScreening

Cloning and Engineering.

The Antibody 16429 DNA sequences encoding immunoglobulin heavy chain(comprising VH1) and light chain (comprising VL1) subunits foranti-huIL-17R antibodies were obtained from Tocker et al. (WO2008/054603 A2) and were cloned using standard recombinant technology.In order to eliminate the binding ability of these antibodies a seriesof site directed mutagenesis clones were generated using polymerasechain reaction (PCR) amplification. The amino acids to be changed wereselected on the basis of location in the complementarity determiningregions (CDRs), change from germline sequence, estimated solventexposure, and aromatic and charge nature. The initial set of mutants wasgermlining and alanine scanning mutants. Subsequently, mutations werecombined and in several cases the alanine scanning mutants were mutatedto introduce negative charge, by replacing the alanine with glutamicacid, or positive charge, by replacing the alanine with arginine.

A representative example of the PCR site direct mutation procedure isthe introduction of an alanine in place of a tryptophan the CDR3 of theanti-IL17 light chain.

PCR amplification was done as a three step process with a 5′ and 3′ PCRused to introduce the mutation and a final overlap PCR to join the twoends of the mutated anti-IL17R light chain. The 5′ PCR use the forwardprimer, 5′-AAG CTC GAG GTC GAC TAG ACC ACC ATG GAA GCC CCA GCG CAG-3′(SEQ ID NO:31) and the reverse primer, 5′-GAA AGT GAG CGG AGC GTT ATCATA CTG CTG ACA-3′ (SEQ ID NO:32). The 3′ PCR use the forward primer,5′-TGT CAG CAG TAT GAT AAC GCT CCG CTC ACT TTC-3′ (SEQ ID NO:33) and thereverse primer, 5′-AAC CGT TTA AAC GCG GCC GCT CAA CAC TCT CCC CTG TTGAA-3′ (SEQ ID NO:34). The overlap PCR use the forward primer, 5′-AAG CTCGAG GTC GAC TAG ACC ACC ATG GAA GCC CCA GCG CAG-3′ (SEQ ID NO:31) andthe reverse primer, 5′-AAC CGT TTA AAC GCG GCC GCT CAA CAC TCT CCC CTGTTG AA-3′ (SEQ ID NO:34).

The PCRs were performed with Phusion HF DNA polymerase (Finnzyme). ThePCR reaction cycles for the 5′ and 3′ PCRs consisted of a 20 seconddenaturation of the anti-IL-17R light chain DNA at 94° C., followed bythree cycles of amplification with each cycle consisting of 20 secondsat 94° C.; 30 seconds at 55° C.; and 30 seconds at 72° C. plus anadditional 27 cycles consisting of 20 seconds at 94° C.; 30 seconds at60° C.; and 30 seconds at 72° C. The reactions were then incubated for 7minutes at 72° C. following the last PCR cycle to insure completeelongation. The PCR reaction cycles for the overlap PCR consisted of a20 second denaturation of the 5′ and 3′ PCR DNAs at 94° C., followed bythree cycles of amplification with each cycles consisting of 20 secondsat 94° C.; 60 seconds at 55° C.; and 40 seconds at 72° C. plus anadditional 27 cycles consisting of 20 seconds at 94° C.; 30 seconds at60° C.; and 40 seconds at 72° C. The reaction was then incubated for 7minutes at 72° C. following the last PCR cycle to insure completeelongation. The overlap PCR product was cloned into pTT5 expressionvector (NRCC) and its sequences determined using ABI DNA sequencinginstrument (Perkin Elmer). Further detail about construct development isfound in Example 5 and Example 6 herein. Table 6 (below) containsdetails about the primers and templates used in cloning the componentsubunits of various embodiments of the inventive immunoglobulins andconjugates, based on the same PCR cycling conditions described in thisparagraph.

Transient Expression to Generate Recombinant Monoclonal Antibodies.

Transient transfections were carried out in HEK 293-6E cells as follows.The human embryonic kidney 293 cell line stably expressing Epstein Barrvirus Nuclear Antigen-1 (293-6E cells) was obtained from the NationalResearch Council (Montreal, Canada). Cells were maintained as serum-freesuspension cultures using F17 medium (Invitrogen, Carlsbad, Calif.)supplemented with 6 mM L-glutamine (Invitrogen, Carlsbad, Calif.), 1.1%F-68 Pluronic (Invitrogen, Carlsbad, Calif.) and 250 μg/ul Geneticin(Invitrigen, Carlsbad, Calif.). The suspension cell cultures weremaintained in Erlenmeyer shake flask cultures. The culture flasks wereshaken at 65 rpm at 37° C. in a humidified, 5% CO₂ atmosphere. A stocksolution (1 mg/ml) of 25-kDa linear PEI (Polysciences, Warrington, Pa.)was prepared in water, acidified with HCl to pH 2.0 until dissolved,then neutralized with NaOH, sterilized by filtration (0.2 μm),aliquoted, and stored at −20° C. until used. Tryptone N1 was obtainedfrom OrganoTechni S.A. (TekniScience, QC, Canada). A stock solution(20%, w/v) was prepared in Freestyle medium (Invitrogem, Carlsbad,Calif.), sterilized by filtration through 0.2 μm filters, and stored at4° C. until use. Typically, transfections were performed at the 1Lscale. Cells (293-6E) were grown too a viable cell density of 1.1×10⁶cells/ml then transfection complexes were prepared in 1/10th volume ofthe final culture volume. For a 1-L transfection culture, transfectioncomplexes were prepared in 100 ml F17 basal medium, and 500 μg plasmidDNA (heavy chain and light chain DNA, 1:1 ratio) was first diluted in100 ml F17 medium. After a 5-minute incubation at room temperature, 1.5ml of PEI solution was added. The complexes were vortexed mildly, thenincubated for 15 minutes at room temperature. The cells were transfectedby adding the transfection complex mix to the cells in the shale flaskculture. 24 hours post-transfection, Tryptone N1 was added to thetransfected culture to a final concentration of 0.5%, and thetransfected cultures were maintained on a shaker at 65 rpm at 37° C. ina humidified, 5% CO₂ atmosphere for another 5 days after which they wereharvested. The conditioned medium was harvested by centrifugation at4000 rpm, and then sterile filtered through 0.2 μm filter (CorningInc.).

Purification of Antibodies.

The transiently expressed antibodies were purified using recombinantprotein A sepharose (GE Healthcare) directly loading the conditionedmedia on the column at 5 ml/min at 7° C. The column was then washed with10 column volumes of Dulbecco's PBS without divalent cations and theneluted with 100 mM acetic acid, pH 3.5. The eluted antibody was pooledbased on the chromatographic profile and the pH was adjusted to 5.0using 2 M Tris base. The pool was then filtered through a 0.8/0.22 μmsyringe filter and then dialyzed against 10 mM acetic acid, 9% sucrose,pH 5.0. The buffer exchanged antibody was then concentrated using aVivaspin 30 kDa centrifugal concentration (Sartorius), and theconcentrated product was filtered through a 0.22 μm cellulose acetatefilter.

BIAcore® Binding Assays.

The lead candidates were then selected based on lack of binding to theIL-17R extracellular domain as determined by BIAcore analysis. Antibody16429 is a human antibody that specifically binds to huIL-17R. Asolution equilibrium binding assay was developed to assess the bindingactivity of a set of antibodies to huIL-17R. Antibody 16429 wasimmobilized to a BIACore® 2000, research grade sensor chip CM5 surfaceaccording to manufacturer's instructions (BIACore, Inc., Piscataway,N.J.). Briefly, carboxyl groups on the sensor chip surfaces wereactivated by injecting 60 μL of a mixture containing 0.2 MN-ethyl-N′-(dimethylaminopropyl) carbodiimide (EDC) and 0.05 MN-hydroxysuccinimide (NHS). Antibody 16429 was diluted in 10 mM sodiumacetate, pH 4.0 and injected over the activated chip surface at 30μL/min for 6 minutes. Excess reactive groups on the surfaces weredeactivated by injecting 60 μL of 1 M ethanolamine. The finalimmobilized level was approximately 6600 resonance units (RU). Asrepresented in FIG. 2A, 10 nM of IL-17R in the absence of solubleantibody was used to establish the 100% binding signal of IL-17R to thefixed 16429 anbibody. To determine antibody binding in solution, 10 nM,100 nM and 1000 nM of the antibody samples were incubated with the 10 nMIL-17R. The decreased binding signal of IL-17R after the antibodyincubation indicates the binding of the antibody to IL-17R in solution.Based on this assay, the 16435, 16438, 16439, 16440, 16441, and 16444antibodies demonstrated substantial reduction in IL-17R bindingcapability. As represented in FIG. 2B, 30 nM IL-17R and 30 μM antibodysamples were used to further demonstrate that the selected antibodieslost their IL-17R binding activity. Based on this assay, all sixantibodies examined (16435, 16438, 16439, 16440, 16441, and 16444)showed no signficant IL-17R binding activity at up to 30 μM antibody.

Cell Based Activity Assay.

Interaction of IL-17 with the IL-17R on cells induces the production ofvarious factors, including growth-related oncogene alpha (GRO-α), fromthese cells. A cell-based characterization assay was developed tomeasure GRO-αreleased using sandwich ELISA. In this ELISA, aGRO-αcapture antibody is utilized to bind GRO-α, and then a biotinylatedGRO-αdetection antibody is used to detect the captured protein.Streptavidin conjugated to horseradish peroxidise (HRP) is then added todetect the amount of biotinylated GRO-αdetection antibody bound. Theamount of HRP bound is measured by evaluation of absorbance at 450 nm.An increase in absorbance at 450 nm is indicative of an increase in theamount of GRO-α produced. In this assay, human foreskin fibroblasts(HFF) are incubated with 5 ng/ml IL-17 and 0.1 μM, 1 μM and 10 μM ofantibody samples. The conditioned cell medium is then harvested andprocessed for assessment of GRO-αlevels using a GRO-αsandwich ELISA. Allsix experimental carrier antibodies (16435, 16438, 16439, 16440, 16441,and 16444) showed no significant blocking activity in this assay at upto 10 μM antibody (FIG. 3).

Analysis of Homogeneity.

Antibodies produced by transient expression were analyzed forhomogeneity using two size exclusion columns (TSK-GEL G3000SWXL, 5 mmparticle size, 7.8×300 mm, TosohBioscience, 08541) in series with a 100mM sodium phosphate, 250 mM NaCl, pH 6.8, mobile phase flowed at 0.5mL/min (FIG. 4A-B). While all the antibodies showed relatively lowlevels of high molecular weight species, 16439 and 16435 had the least,while 16440 had the most. The lead antibodies were further analyzed forproduct quality on a 1.0-mm Tris-glycine 4-20% SDS-PAGE (Novex) usingreducing (FIG. 6) and non-reducing loading buffer (FIG. 5). Allcandidates appeared quite similar by both non-reducing and reducingSDS-PAGE; however, 16433 did show some additional high molecular massmaterial on the reducing SDS-PAGE. Lead candidates were further selectedbased on SEC behavior, SDS-PAGE uniformity, BIAcore binding analysis,cell based assay results and expression levels. Based on these criteria,16435 and 16444 were chosen for further evaluation.

Stable Expression of Antibodies.

Antibody 16435 and 16444 expressing pools were created by transfectingCHO DHFR(−) host cells with corresponding HC and LC expression plasmidset using a standard electroporation procedure. Per each antibodymolecule, 3-4 different transfections were performed to generatemultiple pools. After transfection the cells were grown as a pool in aserum free, (−) GHT (selective growth media to allow for selection andrecovery of the plasmid containing cells. Cell pools grown in (−) GHTselective media were cultured until they reached >85% viability. Theselected cell pools were amplified with 150 nm MTX. When the viabilityof the MTX amplified pools reached >85% viability, the pools werescreened using an abbreviated six day batch production assay with anenriched production media to assess expression. The best pool was chosenbased on the six day assay titer and correct mass confirmation.Subsequently, scale-up production using 11-day fed-batch process wasperformed for the antibody generation, followed by harvest andpurification.

Titers were determined by HPLC assay (FIGS. 7A-B) using a Poros Acolumn, 20 μm, 2.1×30 mm (Applied Biosystems, part #1-5024-12). Briefly,Antibodies in conditioned media were filtered using Spin-X columns(Corning, part #8160) prior to analysis by HPLC, and a blank injectionof 1×PBS (Invitrogen, part #14190-144) was performed prior to injectionof test antibodies and after each analysis run. In addition, conditionedmedia without antibody was injected prior to analysis to condition thecolumn, and new columns were conditioned by triplicate injection of 100μg of control antibody. After a 9-minute wash with PBS at 0.6 ml/min,the antibody was eluted with ImmunoPure IgG Elution Buffer (Pierce, part#21009) and the absorbance at 280 nm was observed. Antibody titers werequantified against a standard plot of control antibody concentrationversus peak area. A control antibody stock was prepared at aconcentration of 4 mg/ml, and five standard antibody concentrations wereprepared by dilution of the antibody control stock in a volume of PBS(0.1 μg/g1 to 1.6 μg/μl). By extending the standard curve, the lowerlimit of detection is 0.02 μg/g1 of antibody, and the higher limit ofquantification is 4 μg/μl. An assumption was made that test antibodieshave similar absorbance characteristics as the control; however titerscan be adjusted by multiplying titer an extinction coefficient ratio ofthe control antibody over the extinction coefficient of the testantibody. The titer assay results show that after scale up to the fedbatch process, the 16435 antibody demonstrated marginally betterexpression than the 16444 carrier antibody.

Purification of Stably Expressed Antibodies.

Stably expressed antibodies were purified by Mab Select Surechromatography (GE Life Sciences) using 8 column volumes of Dulbecco'sPBS without divalent cations as the wash buffer and 100 mM acetic acid,pH 3.5, as the elution buffer at 7° C. The elution peak was pooled basedon the chromatogram, and the pH was raised to about 5.0 using 2 M Trisbase. The pool was then diluted with at least 3 volumes of water,filtered through a 0.22-μm cellulose acetate filter and then loaded onto an SP-HP sepharose column (GE Life Sciences) and washed with 10column volumes of S-Buffer A (20 mM acetic acid, pH 5.0) followed byelution using a 20 column volume gradient to 50% S-Buffer B (20 mMacetic acid, 1 M NaCl, pH 5.0) at 7° C. A pool was made based on thechromatogram and SDS-PAGE analysis, then the material was concentratedabout 6-fold and diafiltered against about 5 volumes of 10 mM aceticacid, 9% sucrose, pH 5.0 using a VivaFlow TFF cassette with a 30 kDamembrane. The dialyzed material was then filtered through a 0.8/0.2-μmcellulose acetate filter and the concentration was determined by theabsorbance at 280 nm. Comparison of the ion exchange chromatographicprofiles of the 16435 and 16444 variants showed no significantdifferences (FIGS. 8A-B).

Analysis of stably expressed antibodies. Analysis of the variants using1.0-mm Tris-glycine 4-20% SDS-PAGE (Novex) with reducing andnon-reducing loading buffer also showed no significant differencebetween the variants (FIGS. 9A-B). However analysis using two sizeexclusion columns (TSK-GEL G3000SWXL, 5 mm particle size, 7.8×300 mm,TosohBioscience, 08541) in series with a 100 mM sodium phosphate, 250 mMNaCl, pH 6.8, mobile phase flowed at 0.5 mL/min showed that 16444possessed more high molecular weight species, and 16435 had a moreprominent pre-peak (FIG. 10).

Antibodies were also analyzed for thermoresistance by DSC using aMicroCal VP-DSC where the samples were heated from 20° C. to 95° C. at arate of 1° C. per minute. DSC directly measures heat changes that occurin biomolecules during controlled increase or decrease in temperature,making it possible to study materials in their native state.

DSC measures the enthalpy (ΔH) of unfolding due to heat denaturation. Abiomolecule in solution is in equilibrium between the native (folded)conformation and its denatured (unfolded) state. The higher the thermaltransition midpoint (Tm), when 50% of the biomolecules are unfolded, themore stable the molecule. DSC is also used to determine the change inheat capacity (ΔCp) of denaturation (see, FIG. 11). The proteins wereincubated at 0.5 mg/ml in 10 mM sodium acetate, 9% sucrose, pH 5.0 (FIG.11). The 16435 antibody produced the most desirable melting profile,with a higher temperature for the secondary transition.

The antibodies were analyzed by reducing and non-reducing CE-SDS (FIG.12A-D). All CE SDS experiments were performed using Beckman PA800 CEsystem (Fullerton, Calif.) equipped with UV diode detector employing 221nm and 220 nm wavelength. A bare-fused silica capillary 50 μm×30.2 cmwas used for the separation analysis. Buffer vial preparation andloading as well as capillary cartridge installation were conducted asdescribed in the Beckman Coulter manual for IgG Purity/Heterogeneity.The running conditions for reduced and non-reduced CE-SDS were similarto those described in Beckman Coulter manual for IgGPurity/Heterogeneity with some modifications which are briefly describedbelow. For non-reducing conditions, the antibody sample (150 μg) wasadded to 20 μl of SDS reaction buffer and 5 μl of 70 mMN-ethylmaleimide. Water was then added to make final volume 35 μl andthe protein concentration was brought to 4.3 mg/ml. The SDS reactionbuffer was made of 4% SDS, 0.01 M citrate phosphate buffer (Sigma) and0.036 M sodium phosphate dibasic. The preparation was vortexedthoroughly, and heated at 45° C. for 5 min. The preparation was thencombined with an additional 115 μl of 4% SDS. After being vortexed andcentrifuged, the preparation was placed in a 200 μL PCR vial and thenloaded onto the PA800 instrument. The sample was injected at the anodewith reverse polarity using −10 kV for 30 sec, and was then separated at−15 kV with 20 psi pressure at both ends of capillary during the 35 minseparation. For reducing conditions, the antibody sample was diluted to2.1 mg/ml by adding purified H₂O, and 95 μl of the antibody was added to105 μL of SDS sample buffer (Beckman) with 5.6% beta mercaptoethanol.The preparation was then vortexed thoroughly and then heated at 70° C.for 10 min. After being centrifuged, the supernatant was placed in a 200μl PCR vial and then loaded onto the PA800 instrument. The sample wasinjected at the anode with reverse polarity using −5 kV for 20 sec, andwas then separated at −15 kV with 20 psi pressure at both ends ofcapillary during 30 min separation. Both 16435 and 16444 antibodiesproduced very similar profiles with both reducing and non-reducingCE-SDS (FIGS. 12A-D).

To measure the light sensitivity of the antibodies, they were incubatedin ambient lab fluorescent lighting or covered in aluminum foil for 3days at room temperature. Light exposed and dark control antibodies werethen analyzed using two size exclusion columns (TSK-GEL G3000SWXL, 5 mmparticle size, 7.8×300 mm, TosohBioscience, 08541) in series with a 100mM sodium phosphate, 250 mM NaCl, pH 6.8, mobile phase flowed at 0.5mL/min. Based on the SEC chromatograms, 16444 showed significantly morelight sensitivity than 16435 (FIG. 13). The antibodies were thenanalyzed by hydrophobic interaction chromatography (HIC) using twoDionex ProPac HIC-10 columns in series with mobile phase A being 1 Mammonium sulfate, 20 mM sodium acetate, pH 5.0 and mobile phase B being20 mM sodium acetate, 5% acetonitrile, pH 5.0. Samples were eluted at0.8 ml/min with a 0-100% linear gradient over 50 minutes observing theabsorbance at 220 nm. Based on the HIC chromatograms, 16435 had anarrower main peak, indicating more product uniformity (FIG. 14). Basedon the lower light sensitivity, better purification yield (1219 mg/L vs.1008 mg/L), better DSC profile, better SEC profile and fewer mutationsfrom the parental antibody, 16435 was chosen as the primary lead forthis family of antibodies.

TABLE 6 PCR primer sets and templates used to clone the indicatedproducts. With Product + Final Template Primer Set Product Primer SetsSEQ ID SEQ ID SEQ ID SEQ ID NOS: NO: NOS: NO: Round One Cloning  (31,32)(33, 34) 187  (31, 34) 189  (35, 37)(38, 36) 304  (35, 36) 322  (35,39)(40, 36) 304  (35, 36) 320  (35, 41)(42, 36) 304  (35, 36) 324 (278,43)(44, 36) 326 (278, 36) 328 (278, 45)(46, 36) 326 (278, 36) 330 RoundTwo Cloning  (31, 213)(214, 34) 181  (31, 34) 185  (31, 215)(216, 34)181  (31, 34) 183  (35, 217)(218, 36) 304  (35, 36) 318  (35, 219)(220,36) 304  (35, 36) 316  (35, 221)(222, 36) 304  (35, 36) 314  (35,223)(224, 36) 304  (35, 36) 312  (35, 225)(226, 36) 304  (35, 36) 310 (35, 227)(228, 36) 304  (35, 36) 308  (35, 229)(230, 36) 304  (35, 36)306 (231, 232)(233, 34) 191 (231, 34) 195 (231, 234)(235, 34) 191 (231,34) 193 (231, 236)(237, 34) 191 (231, 34) 197 (278, 238)(239, 36) 326(278, 36) 332 (278, 240)(241, 36) 326 (278, 36) 334 (278, 242)(243, 36)326 (278, 36) 342 (278, 244)(245, 36) 326 (278, 36) 344 (278, 246)(247,36) 326 (278, 36) 346 (278, 248)(249, 36) 326 (278, 36) 328 (278,250)(251, 36) 326 (278, 36) 330 (278, 252)(253, 36) 326 (278, 36) 348(278, 254)(255, 36) 326 (278, 36) 350 (278, 256)(257, 36) 326 (278, 36)366 (278, 258)(259, 36) 326 (278, 36) 370 Round Three Cloning (doublemutants & germlining) (231, 132)(133, 34) 191 (231, 34) 211 (231,134)(135, 34) 191 (231, 34) 199 (278, 136)(137, 36) 326 (278, 36) 338(278, 138)(139, 36) 326 (278, 36) 372 (278, 140)(141, 36) 326 (278, 36)374 (231, 234)(235, 34) 195 (231, 34) 209 (278, 240)(241, 36) 348 (278,36) 356 (278, 240)(241, 36) 350 (278, 36) 358 Round Four Cloning (chargemutants [A to E or R] and triple mutants) (231, 142)(143, 34) 191 (231,34) 201 (231, 144)(145, 34) 191 (231, 34) 203 (231, 260)(261, 34) 191(231, 34) 205 (231, 262)(263, 34) 191 (231, 34) 207 (278, 264)(265, 36)326 (278, 36) 336 (278, 266)(267, 36) 326 (278, 36) 340 (278, 268)(269,36) 326 (278, 36) 352 (278, 270)(271, 36) 326 (278, 36) 354 (278,272)(273, 36) 326 (278, 36) 360 (278, 274)(275, 36) 326 (278, 36) 362(278, 276)(277, 36) 326 (278, 36) 368 (278, 276)(277, 36) 334 (278, 36)364

Example 2 Generation of Antibodies to Human TRAIL R2 and Screening

Cloning and Engineering.

The Antibody 16449 DNA sequences encoding immunoglobulin heavy chain(comprising VH12) and light chain (comprising VL6) subunits foranti-huTR2 antibodies were obtained from Gliniak et al. (U.S. Pat. No.7,521,048) and were cloned using standard recombinant technology. Inorder to eliminate the binding ability of these antibodies a series ofsite directed mutagenesis clones were generated using polymerase chainreaction (PCR) amplification. The amino acids to be changed wereselected on the basis of location in the complementarity determiningregions (CDRs), change from germline sequence, estimated solventexposure, and aromatic and charge nature. The initial set of mutants wasgermlining and alanine scanning mutants. Subsequently, mutations werecombined and in several cases the alanine scanning mutants were mutatedto introduce negative charge, by replacing the alanine with glutamicacid, or positive charge, by replacing the alanine with arginine.Further detail about construct development is found in Example 5 andTable 6 herein.

Transient Expression to Generate Recombinant Monoclonal Antibodies.

Transient transfections were carried out in HEK 293-6E cells as follows.The human embryonic kidney 293 cell line stably expressing Epstein Barrvirus Nuclear Antigen-1 (293-6E cells) was obtained from the NationalResearch Council (Montreal, Canada). Cells were maintained as serum-freesuspension cultures using F17 medium (Invitrogen, Carlsbad, Calif.)supplemented with 6 mM L-glutamine (Invitrogen, Carlsbad, Calif.), 1.1%F-68 Pluronic (Invitrogen, Carlsbad, Calif.) and 250 μg/ul Geneticin(Invitrigen, Carlsbad, Calif.). The suspension cell cultures weremaintained in Erlenmeyer shake flask cultures. The culture flasks wereshaken at 65 rpm at 37° C. in a humidified, 5% CO₂ atmosphere. A stocksolution (1 mg/ml) of 25-kDa linear PEI (Polysciences, Warrington, Pa.)was prepared in water, acidified with HCl to pH 2.0 until dissolved,then neutralized with NaOH, sterilized by filtration (0.2 μm),aliquoted, and stored at −20° C. until used. Tryptone N1 was obtainedfrom OrganoTechni S.A. (TekniScience, QC, Canada). A stock solution(20%, w/v) was prepared in Freestyle medium (Invitrogem, Carlsbad,Calif.), sterilized by filtration through 0.2 μm filters, and stored at4° C. until use. Typically, transfections were performed at the 1Lscale. Cells (293-6E) were grown too a viable cell density of 1.1×10⁶cells/ml then transfection complexes were prepared in 1/10th volume ofthe final culture volume. For a 1-L transfection culture, transfectioncomplexes were prepared in 100 ml F17 basal medium, and 500 μg plasmidDNA (heavy chain and light chain DNA, 1:1 ratio) was first diluted in100 ml F17 medium. After a 5-minute incubation at room temperature, 1.5ml of PEI solution was added. The complexes were vortexed mildly, thenincubated for 15 minutes at room temperature. The cells were transfectedby adding the transfection complex mix to the cells in the shale flaskculture. 24 hours post-transfection, Tryptone N1 was added to thetransfected culture to a final concentration of 0.5%, and thetransfected cultures were maintained on a shaker at 65 rpm at 37° C. ina humidified, 5% CO₂ atmosphere for another 5 days after which they wereharvested. The conditioned medium was harvested by centrifugation at4000 rpm, and then sterile filtered through 0.2 μm filter (CorningInc.).

Purification of Antibodies.

The transiently expressed antibodies were purified using recombinantprotein A sepharose (GE Healthcare) directly loading the conditionedmedia on the column at 5 ml/min at 7° C. The column was then washed with10 column volumes of Dulbecco's PBS without divalent cations and theneluted with 100 mM acetic acid, pH 3.5. The eluted antibody was pooledbased on the chromatographic profile and the pH was adjusted to 5.0using 2 M tris base. The pool was then filtered through a 0.8/0.22 μmsyringe filter and then dialyzed against 10 mM acetic acid, 9% sucrose,pH 5.0. The buffer exchanged antibody was then concentrated using aVivaspin 30 kDa centrifugal concentrator (Sartorius), and theconcentrated product was filtered through a 0.22 μm cellulose acetatefilter.

BIAcore Binding Assays.

Antibody 16449 is a human antibody that specifically binds to TrailReceptor 2 (TR2). A solution equilibrium binding assay was developed toassess the binding activity of a set of antibodies to TR2. Antibody16449 was immobilized to a CM5 sensor chip surface as described inExample 1 above. Final immobilized level was approximately 8000resonance units (RU). TR2 (1 nM) in the absence of antibody was used toestablish the 100% binding signal of TR2 that is free of antibodybinding in solution. To determine antibody binding in solution, serialdiluted antibody samples in a range of 7 pM to 10 nM were incubated withthe 1 nM TR2. The decreased binding signal of TR2 after the antibodyincubation indicates the binding of the antibody to TR2 in solution. Theresults in FIG. 15 indicate that all three new antibody constructs(16449, 1869, and 1870) retained TR2 binding activity similar to that ofthe original construct.

In other experiments, 10 nM TR2 was incubated with 50 nM and 1 μMantibody samples in the assay as described above. 10 nM TR2 was used todefine the 100% binding signal. Although several of the antibodiesshowed significant lack of binding at 50 nM (16613, 1919, 1913, 1910,1920 and 1922), none showed complete lack of binding at 1000 nM (resultsshown in FIG. 16). Additional point mutagenesis yielded antibodies withlower affinity for TR2 (FIG. 17). Two sites (heavy chain Y125 and lightchain Y53) showed exceptional sensitivity to mutagenesis, particularlywith charged substitutions at position Y125. Double alaninesubstitutions produced variants with even further decreased bindingaffinity for TR2 (FIG. 18). Combining the alanine mutations with chargedmutations in a pairwise, or greater order, fashion produced severalmolecules that did not show significant binding to TR2 even at 10 μMantibody (FIG. 19). From these data, five of the best variants (10186,10184, 4341, 10183, and 4241) were advanced for binding studies at 50 μMantibody (FIGS. 20A-B). All but 10186 showed no significant binding toTR2 even at 50 μM.

Cell Based Activity Assay.

Colo205 is a human colon carcinoma cell line that is sensitive to thepresence of TRAIL. Binding of positive control IgG1 anti-TR2 mAbmolecules (antibody 16449) to TR2 on the surface of Colo205 results incell apoptosis. A Colo205 based cell assay was developed to verify thecell killing efficacy of antibodies. The in vitro biological activity ofthe Antibody 16449 (anti-TRAIL-R2 antibody) is analyzed by its abilityto induce apoptosis in human ascites colorectal adenocarcinoma cell lineColo205. The detection of caspase-3 activation is used as a positivemarker for apoptosis, using the Apo-One™ Homogeneous caspase™-3/7 assaykit (PromegaCorporation, Madison, Wis.), according to the manufacturer'sinstruction. (see, Niles et al., The Apo-One™.Homogeneous Caspase™-3/7assay: a simplified “solution” for apoptosis detection, Cell Notes 2:2-3(2001)). In this method, luminescent caspase-3/7 reagent provides asensitive and robust monitoring of anti-TRAIL-R2 induced caspaseactivation in Colo205 cells. Luminescence produced is proportional tothe amount of caspase activity present. The luminescence of each sampleis measured in a plate-reading luminometer. Biological activity of thetest sample is determined by comparing test sample response to ReferenceStandard response. To compare the samples with standard controlantibody, 200 nM and 10 μM of antibody samples were pre-incubated with 1or 100 μg/ml of protein G. The mixtures were then added to Colo205cultures. FIG. 20C indicates that, unlike the control anti-TR2 mAbmolecules, the antibody samples do not have the ability to kill thecells even at very high concentrations (e.g., 30 μg/mL of antibody).

Analysis of Homogeneity.

The lead antibodies were analyzed for product quality on a 1.0-mmTris-glycine 4-20% SDS-PAGE (Novex) using reducing (FIG. 21B) andnon-reducing loading buffer (FIG. 21A). All candidates appeared quitesimilar by both non-reducing and reducing SDS-PAGE. Antibodies werefurther analyzed for homogeneity using one size exclusion column(Phenomenex SEC3000, 7.8×300 mm) with a 50 mM sodium phosphate, 250 mMNaCl, pH 6.8, mobile phase flowed at 1.0 mL/min (representative resultsare shown in FIG. 22). While all the antibodies showed relatively lowlevels of high molecular weight species, 10185 and 10184 showed slightlymore high molecular mass material. Lead candidates were selected basedon SEC behavior, BIAcore binding analysis, cell based assay results,estimated proteolytic vulnerability and lower shift in the calculatedisoelectric point. Based on these criteria, 4241 and 4341 were chosenfor further evaluation.

Construct Development for Stable Expression.

Pools of stably expressed antibodies 4241 and 4341 were created bytransfecting CHO DHFR(−) host cells with corresponding HC and LCexpression plasmid set using a standard electroporation procedure. Pereach antibody molecule, 3-4 different transfections were performed togenerate multiple pools. After transfection, the cells were grown as apool in a serum free (−)GHT selective growth media to allow forselection and recovery of the plasmid containing cells. Cell pools grownin (−)GHT selective media were cultured until they reached >85%viability. The selected cell pools were amplified with 150 nmmethotrexate (MTX). When the viability of the MTX-amplified poolsreached >85% viability, the pools were screened using an abbreviatedsix-day batch production assay with an enriched production media toassess expression. The best pool was chosen based on the six-day assaytiter and correct mass confirmation. Subsequently, scale-up productionusing 11-day fed-batch process was performed for the antibodygeneration, followed by harvest and purification.

Titers were determined by HPLC assay using a Poros A column, 20 μm,2.1×30 mm (Applied Biosystems, part #1-5024-12). Briefly, Antibodies inconditioned media were filtered using Spin-X columns (Corning, part#8160) prior to analysis by HPLC, and a blank injection of 1×PBS(Invitrogen, part #14190-144) was performed prior to injection of testantibodies and after each analysis run. In addition, conditioned mediawithout antibody was injected prior to analysis to condition the column,and new columns were conditioned by triplicate injection of 100 μg ofcontrol antibody. After a 9-minute wash with PBS at 0.6 ml/min, theantibody was eluted with ImmunoPure IgG Elution Buffer (Pierce, part#21009) and the absorbance at 280 nm was measured.

Antibody titers were quantified against a standard plot of controlantibody concentration versus peak area. A control antibody stock wasprepared at a concentration of 4 mg/ml, and five standard antibodyconcentrations were prepared by dilution of the antibody control stockin a volume of PBS (0.1 μg/μl to 1.6 μg/μl). By extending the standardcurve, the lower limit of detection is 0.02 μg/μl of antibody, and thehigher limit of quantification is 4 μg/μl. An assumption is made thattest antibodies have similar absorbance characteristics as the control;however Titers can be adjusted by multiplying titer an extinctioncoefficient ratio of the control antibody over the extinctioncoefficient of the test antibody. The titer assay results show thatafter scale up to the fed batch process, the 4241 antibody demonstratedmarginally better expression than the 4341 antibody (FIGS. 23A-B).

Purification of Stably Expressed Antibodies.

Stably expressed antibodies were purified by Mab Select Surechromatography (GE Life Sciences) using 8 column volumes of Dulbecco'sPBS without divalent cations as the wash buffer and 100 mM acetic acid,pH 3.5, as the elution buffer at 7° C. The elution peak was pooled basedon the chromatogram, and the pH was raised to about 5.0 using 2 M Trisbase. The pool was then diluted with at least 3 volumes of water,filtered through a 0.22-μm cellulose acetate filter and then loaded onto an SP-HP sepharose column (GE Life Sciences) and washed with 10column volumes of S-Buffer A (20 mM acetic acid, pH 5.0) followed byelution using a 20 column volume gradient to 50% S-Buffer B (20 mMacetic acid, 1 M NaCl, pH 5.0) at 7° C. A pool was made based on thechromatogram and SDS-PAGE analysis, then the material was concentratedabout 6-fold and diafiltered against about 5 volumes of 10 mM aceticacid, 9% sucrose, pH 5.0 using a VivaFlow TFF cassette with a 30 kDamembrane. The dialyzed material was then filtered through a 0.8/0.2-μmcellulose acetate filter and the concentration was determined by theabsorbance at 280 nm. The purification processed samples were analyzedusing a 1.0-mm Tris-glycine 4-20% SDS-PAGE (Novex) reducing loadingbuffer (FIGS. 24A-B). These data showed that both 4241 and 4341antibodies had similar purification characteristics, with no stepsproducing unexpected sample losses.

Analysis of Stably Expressed Antibodies.

Comparison of the ion exchange chromatographic profiles of the 4241 and4341 variants (FIG. 25) showed that 4341 has a narrower main peakindicating less heterogeneity than 4241. Analysis of the variants using1.0-mm Tris-glycine 4-20% SDS-PAGE (Novex) with reducing andnon-reducing loading buffer showed no significant difference between thevariants (FIG. 26A-B). Analysis using two size exclusion columns(TSK-GEL G3000SWXL, 5 mm particle size, 7.8×300 mm, TosohBioscience,08541) in series with a 100 mM sodium phosphate, 250 mM NaCl, pH 6.8,mobile phase flowed at 0.5 mL/min also showed no significant differencebetween the 4241 and 4341 variants (FIGS. 27A-B). The antibodies wereanalyzed for thermoresistance by DSC using a MicroCal VP-DSC where thesamples were heated from 20° C. to 95° C. at a rate of 1° C. per minute.The proteins were at 0.5 mg/ml in 10 mM sodium acetate, 9% sucrose, pH5.0 (FIG. 28). The 4241 antibody produced the most desirable meltingprofile, with a higher temperature for the secondary transition,compared to antibody 4341.

The 4241 and 4341 antibodies were analyzed by reducing and non-reducingCE-SDS (FIGS. 29A-D). All CE SDS experiments were performed usingBeckman PA800 CE system (Fullerton, Calif.) equipped with UV diodedetector employing 221 nm and 220 nm wavelength. A bare-fused silicacapillary 50 μm×30.2 cm was used for the separation analysis. Buffervial preparation and loading as well as capillary cartridge installationwere conducted as described in the Beckman Coulter manual for IgGPurity/Heterogeneity. The running conditions for reduced and non-reducedCE-SDS were similar to those described in Beckman Coulter manual for IgGPurity/Heterogeneity with some modifications which are briefly describedbelow. For non-reducing conditions, the antibody sample (150 μg) wasadded to 20 μl of SDS reaction buffer and 5 μl of 70 mMN-ethylmaleimide. Water was then added to make final volume 35 μl andthe protein concentration was brought to 4.3 mg/ml. The SDS reactionbuffer was made of 4% SDS, 0.01 M citrate phosphate buffer (Sigma) and0.036 M sodium phosphate dibasic. The preparation was vortexedthoroughly, and heated at 45° C. for 5 min. The preparation was thencombined with an additional 115 μl of 4% SDS. After being vortexed andcentrifuged, the preparation was placed in a 200 μl PCR vial and thenloaded onto the PA800 instrument. The sample was injected at the anodewith reverse polarity using −10 kV for 30 sec, and was then separated at−15 kV with 20 psi pressure at both ends of capillary during the 35-minseparation. For reducing conditions, the antibody sample was diluted to2.1 mg/ml by adding purified H₂O, and 95 μl of the antibody was added to105 μl of SDS sample buffer (Beckman) with 5.6% beta mercaptoethanol.The preparation was then vortexed thoroughly and then heated at 70° C.for 10 min. After being centrifuged, the supernatant was placed in a 200μl PCR vial and then loaded onto the PA800 instrument. The sample wasinjected at the anode with reverse polarity using −5 kV for 20 sec, andwas then separated at −15 kV with 20 psi pressure at both ends ofcapillary during 30 min separation. Neither of the antibodies showedsignificant differenced by CE-SDS analysis (FIGS. 29A-D).

Antibodies were also analyzed for homogeneity using high performance ionexchange chromotography (SP-5PW, 10 μm particle, 7.5 mm ID×7.5 cm,TosohBioscience, 08541) using 20 mM acetic acid, pH 5.0 as buffer A and20 mM acetic acid, 1 M NaCl, pH 5.0 as buffer B flowed at 1 mL/min withan 80 minute linear gradient from 0-40% buffer B. Neither purified 4241or 4341 antibody showed significant difference in the high performanceion exchange profiles with this method (FIG. 30). To measure the lightsensitivity of the antibodies, they were incubated in ambient labfluorescent lighting or covered in aluminum foil for 3 days at roomtemperature. The antibodies were then analyzed by hydrophobicinteraction chromatography using two Dionex ProPac HIC-10 columns inseries with mobile phase A being 1 M ammonium sulfate, 20 mM sodiumacetate, pH 5.0 and mobile phase B being 20 mM sodium acetate, 5%acetonitrile, pH 5.0. Samples were eluted at 0.8 ml/min with a 0-100%linear gradient over 50 minutes observing the absorbance at 220 nm.Based on the HIC chromatograms both with and without light exposure,neither antibody displayed significant differences (FIGS. 31A-B). Basedprimarily on the more uniform ion exchange chromatography peak duringpurification 4341 was chosen as the primary lead for this family ofantibodies.

Example 3 Human Tissue Cross-Reactivity Assessment

In general accordance with the guidance laid out in Points to Considerin the Manufacture and Testing of Monoclonal Antibody Products for HumanUse (U.S. Department of Health and Human Services, Food and DrugAdministraton, Center for Biologics Evaluation and Research (1997)), apreliminary non-GLP study was carried out to determine cross-reactivityof inventive antibodies with a variety of human tissues. If an antibodyis intended for drug development, a more extensive testing under GLPconditions is required. The tissue cross-reactivity of antibodies 16435and 4341 was evaluated (Charles River Laboratories, PreclinicalServices, Reno, Nev.) with cryosections of selected human tissues usingAlexa Fluor 488 labeled forms of the test articles. Normal human tissuesfrom two unique individuals (unless otherwise indicated) were obtainedfrom the Special Pathology Services Human Tissue Bank collected by theNational Disease Research Interchange (NDRI, Philadelphia, Pa.),Cureline, Inc. (Burlingame, Calif.), Cybrdi (Rockville, Md.), or RockyMountain Lions Eye Bank (Aurora, Colo.). Tissues tested included humancerebellum, lung, cerebral cortex, ovary (from mature female), eye,placenta, gastrointestinal tract (small intestine), skin (1 individual),heart, spleen, kidney (1 individual), thyroid, liver, testis. Sectionsof fresh-frozen human tissues and control bead blocks (human serumalbumin [HSA] beads) were cut on the cryostat and thaw mounted ontocapillary gap slides. The tissue and control bead slides were fixed incold acetone for approximately 10 minutes at −10° C. to −25° C. Thefixed slides were allowed to dry for at least one hour (to overnight).If stored frozen, fixed slides were removed from the freezer on the dayprior to an experiment and allowed to thaw overnight prior to use. Allthe following steps were performed at room temperature unless otherwisespecified. The slides were incubated with 1× Morphosave™ forapproximately 15 minutes to preserve tissue morphology then washed twotimes for approximately 5 minutes each in1× phosphate-buffered saline(PBS). To block endogenous peroxidase, the slides were incubated in aglucose oxidase solution for approximately 1 hour at approximately 37°C. The slides were washed two times in 1× PBS for approximately 5minutes each. Endogenous biotin was blocked by sequential incubation(approximately 15 minutes each) in avidin and biotin solutions.Following the incubation in biotin, the tissue sections were blockedwith a blocking antibody solution for approximately 25 minutes. AlexaFluor 488-Ab 16435, and Alexa Fluor 488 anti-Ab 4341 were applied tosections at the optimal concentration (2.0 μg/mL) or 5 times the optimalconcentration (10.0 μg/mL) for approximately 25 minutes. Slides werewashed 3 times with wash buffer and then incubated with the secondaryantibody (rabbit anti-Alexa Fluor 488) for approximately 25 minutes.Following incubation with the secondary antibody, slides were washed 4times with wash buffer then incubated with the tertiary antibody(horseradish peroxidase conjugated goat anti-rabbit IgG antibody) forapproximately 25 minutes and binding visualized with a diaminobenzidine(DAB) chromogen substrate. HSA beads were used as a negative control.Tissues were qualified as adequate for immunohistochemistry via stainingwith an antibody against CD31 (anti-CD31) i.e., platelet endothelialcell adhesion molecule (PECAM-1). There was no specific staining in anyhuman tissue examined at either 2.0 or 10.0 μg/mL concentration for anyof the tested antibodies.

Example 4 Pharmacokinetic (PK) Studies of Antibody Embodiments of theInvention in Rats and Cynomolgus Monkeys

The pharmacokinetic profile of the 16435, 16444, 4241, and 4341 carrierantibodies was determined in adult Sprague-Dawley rats (8-12 weeks old)by injecting 5 mg/kg subcutaneously and collecting approximately 250 μLof blood in Microtainer® serum separator tubes at 0, 0.25, 1, 4, 24, 48,72, 96, 168, 336, 504, 672, 840 and 1008 hours post-dose from thelateral tail vein. Each sample was maintained at room temperaturefollowing collection, and following a 30-40 minute clotting period,samples were centrifuged at 2-8° C. at 11,500 rpm for about 10 minutesusing a calibrated Eppendorf 5417R Centrifuge System (BrinkmannInstruments, Inc., Westbury, N.Y.). The collected serum was thentransferred into a pre-labeled (for each rat), cryogenic storage tubeand stored at −60° C. to −80° C. for future analysis. To measure theserum sample concentrations from the PK study samples, the followingmethod was used: ½ area black plate (Corning 3694) was coated with 2μg/ml of anti-hu Fc, antibody 1.35.1 in PBS and then incubated overnightat 4° C. The plate was then washed and blocked with I-Block™ (AppliedBiosystems) overnight at 4° C. If samples needed to be diluted, thenthey were diluted in Rat SD control serum. The standards and sampleswere then diluted 1:20 in I-Block™+5% BSA into 380 μl of dilutingbuffer. The plate was washed and 50-μl samples of pretreated standardsand samples were transferred into an antibody 1.35.1 coated plate andincubated for 1.5 h at room temperature. The plate was washed, then 50μl of 100 ng/ml of anti-hu Fc antibody 21.1-HRP conjugate in I-Block™+5%BSA was added and incubated for 1.5 h. The plate was washed, then 50 μlof Pico substrate were added, after which the plate was immediatelyanalyzed with a luminometer. The pharmacokentic profile was notsignificantly different for any of the four antibodies (FIG. 32) withAUC_(0-t)±SD of 18,492±2,104; 21,021±2,832; 24,045±2,480 and 24,513±972μg/h/mL for antibodies 16435, 16444, 4241 and 4341, respectively.

The pharmacokinetic profile of the 16435 antibody was determined incynomolgus monkeys (Macaca fascicularis) to assess the in vivoparameters. Briefly, a single IV bolus dose of 16435, either 1 mg/kg or10 mg/kg, was administered to mature male cynomolgus monkeys (n=2 pergroup). Serum samples were collected pre-dose and at timepoints 0.25,0.5, 1, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264,288, 312, 360, 408, 456, 504, 552, 600, 648 and 672 hours after antibodyadministration. Samples were assayed for 16435 antibody levels by usingan anti-IgG sandwich ELISA. Time concentration data were analyzed usingnon-compartmental methods with WinNonLin® (Enterprise version 5.1.1,2006, Pharsight® Corp. Mountain View, Calif.). The resultingpharmacokinetic profile did not show any significant abnormalities (FIG.33).

Example 5 Antibody 16435-ShK[1-35, Q16K] Fusion Cloning, Purification &Analysis

Cloning and Expression.

The components of the monovalent 16435-ShK fusion (Antibody 3742)include:

(SEQ ID NO: 109) (a) 16435 kappa LC; (SEQ ID NO: 112) (b) 16435 IgG2 HC;and (SEQ ID NO:377): (c) 16435 IgG2-ShK[1-35, Q16K] SEQ ID NO: 377QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARAQLYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC//.

The desired product antibody fusion (3742) was a full antibody with theShK[1-35, Q16K] peptide (SEQ ID NO:76) fused to the C-terminus of oneheavy chain (see, schematic representation in FIG. 34). With twodifferent heavy chains sharing one variety of light chain, the ratio ofheavy chain:light chain:heavy chain-ShK[1-35, Q16K] was 1:2:1. Theexpected expression products are 16435 IgG2, monovalent 16435IgG2-ShK[1-35, Q16K], and divalent 16435 IgG2-ShK[1-35, Q16K]. Themonovalent 16435 IgG2-Shk fusion protein was isolated from the mix usingcation exchange chromatography, as described herein.

The ShK[1-35, Q16K] fragment was generated using constructpTT5-aKLH120.6-IgG2-HC-L10-ShK[1-35, Q16K], encoding (SEQ ID NO:389), asa template, which was digested with Stul and NotI and purified with thePCR Purification Kit (Qiagen). At the same time, pDC324 (SEQ ID NO:111)was digested with Stul and NotI, treated with Calf Intestine Phosphatase(CIP) and run out on a 1% agarose gel. The larger fragment was cut outand gel purified by Qiagen's Gel Purification Kit. The purifiedShk[1-35, Q16K] fragment was ligated to the large vector fragment andtransformed into OneShot Top10 bacteria. DNAs from transformed bacterialcolonies were isolated and submitted for sequencing. Although analysisof several sequences of clones yielded a 100% percent match with theabove sequence, only one clone was selected for large scaled plasmidpurification. The final pDC324-16435-IgG2-HC-L10-ShK[1-35, Q16K]construct encoded an IgG2-HC-L10-ShK[1-35, Q16K] fusion polypeptide (SEQID:377).

Purification.

Initial purification of the 3742 conditioned media was done by affinityFPLC capture of the Fc region using Protein A Sepharose (GE Healthcare)followed by a column wash with Dulbecco's PBS without divalent cations(Invitrogen) and step elution with 100 mM acetic acid, pH 3.5. Proteincontaining fractions were pooled and neutralized to pH 5.0 with 10 NNaOH and diluted 5-times volume with water. The material was filteredthrough a 0.45 μm cellulose acetate filter (Corning) and furtherpurified by cation exchange FPLC (SP Sepharose High Performance; GEHealthcare). Samples were loaded onto a column equilibrated with 100%buffer A (50 mM acetic acid, pH 5.0) and eluted with a gradient of 0 to800 mM NaCl over 30 column volumes. Peaks containing monovalent specieswere pooled and formulated into 10 mM sodium acetate, 9% sucrose, pH5.0.

Analysis.

Reducing and non-reducing SDS-PAGE analysis was done on 3742 pools using4-12% tris-glycine gels (Invitrogen) with 2 μg of protein, stained withQuickBlue (Boston Biologicals). Based on the SDS-PAGE there were nosignificant differences between the pools (FIG. 35). Analytical SEC wasdone using a Biosep SEC-S3000 column (Phenomenex) with an isocraticelution using 50 mM sodium phosphate, 250 mM NaCl, pH 6.9 as the mobilephase at 1 ml/min (FIGS. 36A-D). All four pools showed relatively lowlevels of aggregate based on the SEC data; however, pool 1 showedsomewhat higher levels than the other pools.

The final 3742 samples were characterized by LC-MS analysis of reducedheavy chain (FIGS. 38A-D) and light chain (FIGS. 37A-D). The product waschromatographed through a Waters MassPREP micro desalting column using aWaters ACQUITY UPLC system. The column was set at 80° C. and the proteineluted using a linear gradient of increasing acetonitrile concentrationin 0.1% formic acid. The column effluent was directed into a Waters LCTPremier ESI-TOF mass spectrometer for mass analysis. The instrument wasrun in the positive V mode. The capillary voltage was set at 3,200 V andthe cone voltage at 80 V. The mass spectrum was acquired from 800 to3000 m/z and deconvoluted using the MaxEnt1 software provided by theinstrument manufacturer. All four pools yielded the expected mass withinthe error of the instrument, indicating all pools were producing theexpected product (FIGS. 37A-D and FIGS. 38A-D).

Whole Blood Assay.

An ex vivo assay was employed to examine impact of toxin peptide analogKv1.3 inhibitors on secretion of IL-2 and IFN-γ. The potency of ShKanalogs and conjugates in blocking T cell inflammation in human wholeblood was examined using an ex vivo assay that has been describedearlier (see Example 46 of WO 2008/088422 A2, incorporated herein byreference in its entirety). In brief, 50% human whole blood wasstimulated with thapsigargin to induce store depletion, calciummobilization and cytokine secretion. To assess the potency of moleculesin blocking T cell cytokine secretion, various concentrations of Kv1 0.3blocking peptides and peptide-conjugates were pre-incubated with thehuman whole blood sample for 30-60 min prior to addition of thethapsigargin stimulus. After 48 hours at 37° C. and 5% CO₂, conditionedmedium was collected and the level of cytokine secretion was determinedusing a 4-spot electrochemiluminescent immunoassay from MesoScaleDiscovery. Using thapsigargin stimulus, the cytokines IL-2 and IFN-gwere secreted robustly from blood isolated from multiple donors. TheIL-2 and IFN-g produced in human whole blood following thapsigarginstimulation were produced from T cells, as revealed by intracellularcytokine staining and fluorescence-activated cell sorting (FACS)analysis. Kv1 0.3 is the major voltage-gated potassium channel presenton T cells. Allowing for K⁺ efflux, Kv1 0.3 provides the driving forcefor continued Ca^(t)′ influx which is necessary for the sustainedelevation in intracellular calcium needed for efficient T cellactivation and cytokine secretion. Kv1 3 inhibitors have been shownearlier to suppress this calcium flux induced by TCR ligation (G. C. Kooet al., 1999, Cell. Immunol. 197, 99-107). Thapsigargin-inducedstore-depletion and TCR ligation elicits similar patterns of Ca²⁺mobilization in isolated T cells (E. Donnadieu et al., 1991, J. Biol.Chem. 267, 25864-25872), but we have found thapsigargin gives a morerobust response in whole blood. Therefore, we employed a bioassaywhereby the bioactivity of Kv1 3 inhibitors is assessed by examiningtheir ability to block thapsigargin-induced cytokine secretion from Tcells in human whole blood. Since whole blood is a complex fluidcontaining high protein levels, the activity of peptides and peptideconjugates in this whole blood assay has an additional advantage inassessing the molecules stability over 48 hours in a biologicallyrelevant fluid. The whole blood assay provides important confirmation ofthe Kv1 0.3 potency of molecules determined by electrophysiology(ePhys), since ePhys assays are generally of short duration (<1-2 hours)and use physiological saline containing no protein. The longer durationof the whole blood assay may allow for more effective determination ofequilibrium binding kinetics relative to ePhys studies which are ofshort duration. As seen in Table 7A (below), all four pools of3742-ShK(1-35, Q16K) showed good potency in the human whole blood assay,indicating the isolated molecules have obtained the proper tertiarystructure and are reasonably stable in serum for 48 hours. Table 7B(below) shows that the potency was comparable to other ShK-conjugatedmolecules.

TABLE 7A Human whole blood (“WB”) assays of four pools of 3742 (SEQ IDNOS: 377; 109, 112; 109) of IL-2 and interferon-gamma (“IFNγ”) wereconducted as described in Example 5 herein. IC₅₀ IFNγ IC₅₀ IL-2 Pool(pM) (pM) 1 708 2220 2 599 2461 3 598 1649 4 412 909

TABLE 7B Data demonstrating potency of various conjugates of [Lys16]ShKin the Whole Blood Assay. Toxin peptides and toxin peptide analogs werePEGylated as described in Example 9 herein. Immunoglobulin-containingcompounds were recombinantly expressed and purified as described inExample 8. Human whole blood (“WB”) assays of IL-2 and interferon-gamma(“IFNg”) were conducted as described in Example 5 herein). WB WB Potency(IL-2) (IFNg) Relative SEQ ID NO or Conjugate IC50 IC50 to ShK citationType Designation (nM) (nM) (WB, IL2) 378 none ShK(1-35) 0.067 0.078 1.00 76 none [Lys16]ShK 0.110 0.158 1.64 379 none [Lys16]ShK-Ala 0.138 0.2662.06 380 PEG 20 kDa-PEG-ShK 0.380 0.840 5.67 381 PEG 20 kDa-PEG- 0.0920.160 1.37 [Lys16]ShK 382 PEG 20 kDa-PEG- 0.754 1.187 11.25[Lys16]ShK-Ala 377; 109; 112; 109 IgG2 Monovalent antibody 0.412 0.9096.15 # 3742-ShK(1-35, Q16K), Pool 4 Example 1, IgG1 Bivalent Fc-L10-0.386 0.320 5.76 WO2008/088422 A2 ShK[1-35] homodimer Example 2, IgG1Bivalent Fc-L10- 0.585 2.285 8.73 WO2008/088422 A2 ShK[2-35] homodimerExample 2, IgG1 Monovalent Fc/Fc- 2.149 5.199 32.07 WO2008/088422 A2L10-ShK[2-35] heterodimer  1; 26 IgG2 Monovalent Fc/Fc- 0.160 0.499 2.39ShK(1-35 Q16K) heterodimer 26; 26 IgG2 Bivalent Fc-ShK(1- 1.850 3.14027.61 35, Q16K) homodimer

Example 6 Ab 4341-ShK(1-35, Q16K), 4341-FGF21 and 16435-FGF21 FusionConstruct Generation

Antibody 16435-huFGF21 Fusion (Ab 10162).

The components of the 16435-huFGF21 fusion include:

(SEQ ID NO: 109) (a) 16435 kappa LC; (R118A; SEQ ID NO: 112)(b) 16435 HC; and (SEQ ID NO: 384): (c) 16435 IgG2-HC-huFGF21 [1-181]SEQ ID NO: 384 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARAQLYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGGSGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS//.

The 16435 huIgG2-HC-L15-huFGF21 [1-181] fragment was generated usingconstruct pTT5-aKLH120.6-IgG2-HC-L15-huFGF21 [1-181] (SEQ ID NO:130) asa template, which was digested with BsmBI and NotI and purified with theQiagen Gel Purification Kit. At the same time, pTT5-16435 IgG2 HC wasdigested with BsmBI and NotI, and run out on a 1% agarose gel. Thevector fragments, which contained the 16435 heavy chain variable region,were cut out and gel purified by Qiagen Gel Purification Kit. Thepurified huIgG2-HC-L15-huFGF21 [1-181] fragment was ligated to thevector fragments containing the 16435 heavy chain variable region andtransformed into DH10b bacteria. DNAs from transformed bacterialcolonies were isolated and submitted for sequencing. Although analysisof several sequences of clones yielded a 100% percent match with theabove sequence, only one clone was selected for large scaled plasmidpurification. The final pTT5-16435-IgG2-HC-L15-huFGF21 [1-181] constructencoded an IgG2-HC-L15-huFGF21 [1-181] fusion polypeptide (SEQ ID:384)

Antibody 4341-huFGF21 Fusion (Ab 10163).

The components of the 4341-ShK[1-35, Q16K] fusion (Ab 10163) include:

(SEQ ID NO: 115) (a) 4341 kappa LC; (Y125A; SEQ ID NO: 118) (b) 4341 HC;and (SEQ ID NO: 386): (c) 4341 IgG2-HC-huFGF21 [1-181] SEQ ID NO: 386QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLRLSSVTAADTAVYYCARDRGGDYAYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGGSGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS//.

The huIgG2-HC-L15-huFGF21 [1-181] fragment was generated using constructpTT5-aKLH120.6-IgG2-HC-L15-huFGF21 [1-181] as a template, which wasdigested with BsmBI and NotI and purified with the Qiagen GelPurification Kit. At the same time, pTT5-4341 IgG2 HC was digested withBsmBI and NotI, and run out on a 1% agarose gel. The larger fragment,which contained the 4341 heavy chain variable region, was cut out andgel purified by Qiagen Gel Purification Kit. The purifiedhuIgG2-HC-L15-huFGF21 [1-181] fragment was ligated to the large vectorfragment containing the 4341 heavy chain variable region and transformedinto DH10b bacteria. DNAs from transformed bacterial colonies wereisolated and submitted for sequencing. Although analysis of severalsequences of clones yielded a 100% percent match with the abovesequence, only one clone was selected for large scaled plasmidpurification. The final pTT5-4341-IgG2-HC-L15-huFGF21 [1-181] constructencoded an IgG2-HC-L15-huFGF21 [1-181] fusion polypeptide (SEQ ID:386).

4341-ShK[1-35, Q16K] Fusion (Antibody 10164).

The components of the 4341-ShK[1-35, Q16K] fusion (Ab 10164) include:

(SEQ ID NO: 115) (a) 4341 kappa LC; (Y125A; SEQ ID NO: 118) (b) 4341 HC;and (SEQ ID NO: 388): (c) 4341 IgG2-HC-ShK [1-35, Q16K] SEQ ID NO: 388QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLRLSSVTAADTAVYYCARDRGGDYAYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPGGGGGSGGGGSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC//.

The huIgG2-HC-L10-ShK [1-35, Q16K] fragment was generated usingconstruct pDC324-16435-HC-L10-IgG2-ShK [1-35, Q16K] (SEQ ID NO:376) as atemplate, which was digested with BsmBI and NotI and purified with theQiagen Gel Purification Kit. At the same time, pTT5-4341 IgG2 HC wasdigested with BsmBI and NotI, and run out on a 1% agarose gel. Thelarger fragment, which contained the 4341 heavy chain variable region,was cut out and gel purified by Qiagen Gel Purification Kit. Thepurified huIgG2-HC-L10-ShK [1-35, Q16K] fragment was ligated to thelarge vector fragment containing the 4341 heavy chain variable regionand transformed into DH10b bacteria. DNAs from transformed bacterialcolonies were isolated and submitted for sequencing. Although analysisof several sequences of clones yielded a 100% percent match with theabove sequence, only one clone was selected for large scaled plasmidpurification. The final pTT5-4341-IgG2-HC-L10-ShK [1-35, Q16K] constructencoded an IgG2-HC-L10-ShK [1-35, Q16K] fusion polypeptide (SEQ IDNO:388).

Example 7 Ab 4341-ShK, 4341-FGF21 and 16435-FGF21 Fusion Expression,Purification & Analysis

Transient transfections were carried out in HEK 293-6E cells as follows.The human embryonic kidney 293 cell line stably expressing Epstein Barrvirus Nuclear Antigen-1 (293-6E cells) was obtained from the NationalResearch Council (Montreal, Canada). Cells were maintained as serum-freesuspension cultures using F17 medium (Invitrogen, Carlsbad, Calif.)supplemented with 6 mM L-glutamine (Invitrogen, Carlsbad, Calif.), 1.1%F-68 Pluronic (Invitrogen, Carlsbad, Calif.) and 250 μg/ul Geneticin(Invitrogen, Carlsbad, Calif.). The suspension cell cultures weremaintained in Erlenmeyer shake flask cultures. The culture flasks wereshaken at 65 rpm at 37° C. in a humidified, 5% CO₂ atmosphere. A stocksolution (1 mg/ml) of 25-kDa linear PEI (Polysciences, Warrington, Pa.)was prepared in water, acidified with HCl to pH 2.0 until dissolved,then neutralized with NaOH, sterilized by filtration (0.2 μm),aliquoted, and stored at −20° C. until used. Tryptone N1 was obtainedfrom OrganoTechni S.A. (TekniScience, QC, Canada). A stock solution(20%, w/v) was prepared in F17 medium, sterilized by filtration through0.2 μm filters, and stored at 4° C. until use. Typically, transfectionswere performed at the 1L scale. Cells (293-6E) were grown too a viablecell density of 1.1×10⁶ cells/ml then transfection complexes wereprepared in 1/10th volume of the final culture volume. For a 1-Ltransfection culture, transfection complexes were prepared in 100 ml F17basal medium, and 500 μg plasmid DNA (heavy chain and light chain DNA,1:1 ratio) was first diluted in 100 ml F17 medium. After a 5-minuteincubation at room temperature, 1.5 ml of PEI solution was added. Thecomplexes were vortexed mildly, then incubated for 15 minutes at roomtemperature. The cells were transfected by adding the transfectioncomplex mix to the cells in the shake flask culture. 24 hourspost-transfection, Tryptone N1 was added to the transfected culture to afinal concentration of 0.5%, and the transfected cultures weremaintained on a shaker at 65 rpm at 37° C. in a humidified, 5% CO₂atmosphere for another 5 days after which they were harvested. Theconditioned medium was harvested by centrifugation at 4000 rpm, and thensterile filtered through 0.2 μm filter (Corning Inc.).

The transiently expressed antibodies were purified using recombinantprotein A sepharose (GE Healthcare) directly loading the conditionedmedia on the column at 5 ml/min at 7° C. The column was then washed with10 column volumes of Dulbecco's PBS without divalent cations and theneluted with 100 mM acetic acid, pH 3.5. The eluted antibodies werepooled based on the chromatographic profile and the pH was adjusted to5.0 using 2 M tris base. The pools were then filtered through a 0.8/0.22um syringe filter and then dialyzed against 10 mM acetic acid, 9%sucrose, pH 5.0. The buffer exchanged antibodies were then concentratedusing a Vivaspin 30 kDa centrifugal concentration (Sartorius), and theconcentrated products were filtered through a 0.22 um cellulose acetatefilter. All conditioned media, including a mock transfection, wereanalyzed using a 1.0 mm Tris-glycine 4-20% SDS-PAGE run at 35mA/1000V/250 W for 55 min (FIG. 39A) loading 10 μl conditioned media.The band above the 250 molecular weight marker not observed in the mocktransfection sample is likely the expressed product. All threeexperimental transfections showed a significant quantity of the expectedproduct on the SDS-PAGE.

Antibody fusions were analyzed for product quality on a 1.0-mmTris-glycine 4-20% SDS-PAGE (Novex) using reducing and non-reducingloading buffer (FIG. 39B). All candidates electrophoresed as expected byboth non-reducing and reducing SDS-PAGE; however, 10162 and 10163 showsome slower migrating than expected bands, possibly indicating partialglycosylation. Antibodies were further analyzed for homogeneity usingone size exclusion column (Phenomenex SEC3000, 7.8×300 mm) with a 50 mMsodium phosphate, 250 mM NaCl, pH 6.8, mobile phase flowed at 1.0 mL/min(FIG. 40). The 10162 and 10163 fusions eluted as expected and showedrelatively low levels of high molecular weight species; however, the10164 fusion eluted earlier than expected, possibly indicatingaggregation.

LC-MS analysis was conducted of reduced light chain (FIGS. 41A-C) andheavy chain (FIGS. 42A-C), respectively, of the final 4341-ShK,4341-FGF21, and 16435-FGF21 samples. The FGF21 fusion samples weredeglycosylated prior to reduction using the PNGase F technique asdescribed by the manufacturer (QA Bio, LLC, Palm Desert, Calif.), exceptthat the substrate to enzyme ratio was 10 μg substrate to 1 μL enzyme.The product was chromatographed through a Zorbax SB300 C8 50×1 mm 3micron column using an Agilent 1100 capillary HPLC system. The columnwas set at 75° C. and the protein eluted using a gradient of increasingn-propanol concentration in 0.1% trifluoroacetic acid. The columneffluent was directed into an Agilent-TOF mass spectrometer for massanalysis. The capillary voltage was set at 3,200 V and the fragmentorvoltage at 225 V. The mass spectrum was acquired from 800 to 3000 m/zand deconvoluted using the MassHunter software provided by theinstrument manufacturer. All samples possessed the expected mass withinthe error of the instrument, indicating all pools contained the expectedproduct.

Example 8 Expression and Purification of Monovalent or MultivalentImmunoglobulin- and/or Fc Domain-Toxin Peptide Analog Fusions

An assortment of monovalent, bivalent and trivalent structures wereexpressed and purified for comparison, including exemplary embodimentsof the invention, as illustrated in Table 7B in Example 5. Thoseincluded antibody IgG2- or IgG1-ShK fusion variants (see FIG. 1F-L). Forexample, bivalent Fc-L10-ShK[1-35], monovalent immunoglobulin heavychain-[Lys16]ShK fusion antibody; see FIG. 1F). IgG2 Fc/Fc-ShK variants(see FIG. 1A), bivalent Fc-L10-ShK[2-35], monovalent Fc/Fc-L10-ShK[2-35]were also made for comparison, by recombinant methods as described inSullivan et al., WO 2008/088422 A2, and in particular Examples 1, 2, and56 therein, incorporated by reference in its entirety, or as modifiedherein.

Transient expression system used to generate toxin peptide analog-Fcfusions (“peptibodies”) or other immunoglobulin fusion embodiments. HEK293-6E cells were maintained in 3 L Fernbach Erlenmeyer Flasks between2e5 and 1.2e6 cells/ml in F17 medium supplemented with L-Glutamine (6mM) and Geneticin (25 μg/ml) at 37° C., 5% CO₂, and shaken at 65 RPM. Atthe time of transfection, cells were diluted to 1.1×10⁶ cells/mL in theF17 medium mentioned above at 90% of the final culture volume. DNAcomplex was prepared in Freestyle293 medium at 10% of the final culturevolume. DNA complex includes 500 ug total DNA per liter of culture and1.5 ml PEImax per liter of culture. DNA complex is briefly shaken onceingredients are added and incubated at room temperature for 10 to 20minutes before being added to the cell culture and placed back in theincubator. The day after transfection, Tryptone N1 (5 g/L) was added tothe culture from liquid 20% stock. Six days after transfection, culturewas centrifuged at 4,000 RPM for 40 minutes to pellet the cells and thecultured medium was harvested through a 0.45 um filter.

In preparing the DNA complex, the ratio of plasmids was proportional tothe desired molar ratio of the peptides needed to generate the intendedproduct. The components of the IgG2 Fc/Fc-ShK include IgG2 Fc and IgG2Fc-ShK at a 1:1 ratio. During expression these assemble into IgG2 Fchomodimers, IgG2 Fc/Fc-ShK heterodimers, and IgG2 Fc-ShK homodimers. TheIgG2 Fc/Fc-ShK heterodimer (monovalent form) was isolated duringpurification using cation exchange chromatography.

IgG2 Fc-ShK[2-35]; IgG2 Fc Shk[2-35, Q16K]; IgG2 Fc-Shk[1-35]; IgG2Fc-ShK[1-35, Q16K] mammalian expression. DNA sequences coding for theimmunoglobulin Fc domain of human IgG2:

SEQ ID NO: 1 MEWSWVFLFFLSVTTGVHSERKVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//,fused in-frame to a monomer of the Kv1 3 inhibitor peptide ShK[2-35] ora mutated ShK[2-35, Q16K] were constructed using standard PCRtechnology. The ShK[2-35] or ShK[2-35, Q16K] and the 10 amino acidlinker portion of the molecule were generated in a PCR reaction usingthe original Fc-2xL-ShK[2-35] in pcDNA3.1(+)CMVi as a template (seeSullivan et al., WO 2008/088422 A2, Example 2, FIGS. 15A-B therein). TheShK[1-35] was generated in a PCR reaction using the originalFc-2xL-ShK[1-35] in pcDNA3.1(+)CMVi as a template (Sullivan et al., WO2008/088422 A2, Example 1, FIGS. 14A-B therein). These ShK constructshave the following modified VH21 Signal peptide amino acid sequence ofMEWSWVFLFFLSVTTGVHS// SEQ ID NO:2 generated from apSelexis-Vh21-hIgG2-Fc template with the following oligos:

(SEQ ID NO: 3) 5′- CAT GAA TTC CCC ACC ATG GAA TGG AGC TGG- 3′; and(SEQ ID NO: 4) 5′- CA CGG TGG GCA CTC GAC TTT GCGCTC GGA GTG GAC ACC -3′.

Wild Type ShK[2-35] with N-terminal linker extension (amino acidsequence GGGGSGGGGSSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC// SEQ ID NO:6) wasencoded by the DNA sequence below:GGAGGAGGAGGATCCGGAGGAGGAGGAAGCAGCTGCATCGACACCATCCCCAAGAGCCGCTGCACCGCCTTCCAGTGCAAGCACAGCATGAAGTACCGCCTGAGCTTCTGCCGCAAGACCTGCGGCACCTGC// SEQ ID NO:5. A fragment containingthis coding sequence (SEQ ID NO:5) was generated using the oligos below(SEQ ID NO:7 and SEQ ID NO:8)- and the original Fc-L10-ShK[2-35] inpcDNA3.1(+)CMVi as a template (Sullivan et al., WO 2008/088422 A2,Example 2, FIGS. 15A-B therein, incorporated by reference):

(SEQ ID NO: 7) 5′-GTC CAC TCC GAG CGC AAA GTC GAG TGC CCA CCG TGC C-3′;and (SEQ ID NO: 8) 5′- TCC TCC TCC TTT ACC CGG AGA CAG GGA GAG -3′//.

Mutant ShK[2-35, Q16K] was generated using site directed mutagenesiswith Stratagene's QuikChange Multi site-Directed Mutagenesis kitcat#200531 per the manufacterer's instruction. Oligos used to generatethe mutagenesis were:

5′-GCT GCA CCG CCT TCA AGT GCA AGC ACA GC 3′ (SEQ ID NO:9); and

5′-GCT GTG CTT GCA CTT GAA GGC GGT GCA GC-3′ (SEQ ID NO:10); and usingthe original Fc-L10-ShK[2-35] in pcDNA3.1(+)CMVi as a template (Sullivanet al., WO 2008/088422 A2, Example 2, FIGS. 15A-B therein) resulting inthe DNA coding sequence

(SEQ ID NO: 11) Ggaggaggaggatccggaggaggaggaagcagctgcatcgacaccatccccaagagccgctgcaccgccttcaagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgc//,which encodes the amino acid sequence Shk(2-35, K16) with a N-terminallinker extension:

SEQ ID NO: 12) ggggsggggsscidtipksrctafkckhsmkyrlsfcrktcgtc//.

ShK[1-35]WT fragment was generated using the original Fc-2xL-ShK[1-35]in pcDNA3.1(+)CMVi as a template (Sullivan et al., WO 2008/088422 A2,Example 1, FIGS. 14A-B therein) and oligos:

(SEQ ID NO: 7) 5′-GTC CAC TCC GAG CGC AAA GTC GAG TGC CCA CCG TGC C-3′;and (SEQ ID NO: 8) 5′- TCC TCC TCC TTT ACC CGG AGA CAG GGA GAG -3′.

The IgG2Fc region was generated using oligos:

5′-CCG GGT AAA GGA GGA GGA GGA TCC GGA G-3′ (SEQ ID NO:13); and

5′-CAT GCG GCC GCT CAT TAG CAG GTG-3′ (SEQ ID NO:14), and the pSelexisVh21-hIgG2-Fc template resulting in a fragment containing the followingDNA coding sequence:

SEQ ID NO: 15 gcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa a//,which encodes the amino acid sequence SEQ ID NO: 16)appvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhea lhnhytqkslslspgk.

The PCR fragments were generated and the products were run out on a gel.After gel purification, the DNA fragments were put together in a PCRtube and sewn together with outside primers:

(SEQ ID NO: 3) 5′- CAT GAA TTC CCC ACC ATG GAA TGG AGC TGG -3′; and(SEQ ID NO: 14) 5′- CAT GCG GCC GCT CAT TAG CAG GTG - 3′.

The PCR products were digested with EcoRI and NotI (Roche) restrictionenzymes and agarose gel purified by Gel Purification Kit. At the sametime, the pTT14 vector (an Amgen vector containing a CMV promoter, PolyA tail and a Puromycin resistance gene) was digested with EcoRI and NotIrestriction enzymes and the large fragment was purified by GelPurification Kit. Each purified PCR product was ligated to the largefragment and transformed into OneShot Top10 bacteria. DNAs fromtransformed bacterial colonies were isolated and subjected to EcoRI andNotI restriction enzyme digestions and resolved on a one percent agarosegel. DNAs resulting in an expected pattern were submitted forsequencing. Although, analysis of several sequences of clones yielded a100% percent match with the above sequence, only one clone of eachconstruct was selected for large scaled plasmid purification. The finalpTT14-VH1SP-IgG2-Fc construct encoded IgG2-Fc-L10-ShK(2-35) fusionpolypeptide having the following sequence:

(SEQ ID NO: 17) Mewswvflfflsvttgvhserkvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsscidtipksrctafqckhsmkyrlsfcrktcgtc//.

The pTT14-VH21SP-IgG2-Fc-L10-ShK(2-35,Q16K) construct encoded a IgG2-FcL10-ShK(2-35, Q16K) fusion polypeptide sequence:

SEQ ID NO: 18 MewswvflfflsvttgvhserkvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsscidtipksrctafKckhsmkyrlsfcrktcgtc//;

and pTT14-VH21SP-IgG2-Fc ShK1-35 construct contained a coding sequencefor IgG2 Fc-L10-ShK(1-35) fusion polypeptide having the followingsequence:

(SEQ ID NO: 19) mewswvflfflsvttgvhserkvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsrscidtipksrctafqckhsmkyrlsfcrktcgtc//.

Generating the VH21 SP-IgG2-Fc-only construct in pYD 16 (an Amgen vectorcontaining a CMV promoter, Poly A tail and a Hygromycin resistance gene)occurred as follows: The VH21 signal peptide was generated using thefollowing oligos:

5′-CAT AAG CTT CCC ACC ATG GAA TGG AGC TGG-3′ (SEQ ID NO:20); and

5′-CA CGG TGG GCA CTC GAC TTT GCG CTC GGA GTG GAC ACC-3′ (SEQ ID NO:4),and using the pSelexis template as noted above.

The Fc region was generated using the pSelexis template described aboveand following oligos:

(SEQ ID NO: 7) 5′-GTC CAC TCC GAG CGC AAA GTC GAG TGC CCA CCG TGC C-3′;and (SEQ ID NO: 21) 5′- CAT GGA TCC TCA TTT ACC CGG AGA CAG GGA G -3′.

The PCR fragments were gel purified and sewn together in single PCRreaction using outside primers GGT TGA GAG GTG CCA GAT GTC AGG GCT GCAGCA GCG GC// SEQ ID NO:391 and CAG CTG CAC CTG ACC ACC ACC TCC ACC GCTATG CTG AGC GCG// SEQ ID NO:392. The resulting PCR fragment was gelpurified, and digested by HindIII and BamHI. Concurrently, pYD16 vector(an Amgen vector containing a CMV promoter, Poly A tail and a Hygromycinresistance gene) was also cut by HindIII and BamHI and the large vectorfragment was purified by Qiagen's Gel Purification Kit. The purified PCRproduct was ligated to the large fragment and transformed into OneShotTop10 bacteria. DNA from transformed bacterial colonies were isolatedand subjected to HindIII and BamHI restriction enzyme digestions andresolved on a one percent agarose gel. DNAs resulting in an expectedpattern were submitted for sequencing. Although, analysis of severalsequences of clones yielded a 100% percent match with the abovesequence, only one clone was selected for large scaled plasmidpurification. The final pYD16-VH21SP-IgG2-Fc construct encoded humanIgG2-Fc (SEQ ID NO:1 above).

IgG2-Fc ShK[1-35, Q16K] Mammalian Expression.

Using the DNA pTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK[1-35, Q16K]construct, the fragment containing the DNA coding sequence

(SEQ ID NO: 22) ggatccggaggaggaggaagccgcagctgcatcgacaccatccccaagagccgctgcaccgccttcaagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgctaatgagcggccgctcgaggccgg caaggccggatcc//

was cut out using BamHI/BamHI. This coding sequence (SEQ ID NO:23)encodes ShK(1-35, Q16K) with an N-terminal linker sequence:

(SEQ ID NO: 23) GSGGGGSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC//.

At the same time, pTT14-hIgG2-Fc-ShK[1-35]WT construct, was alsodigested by BamHI/BamHI, thereby removing the Shk[1-35] coding region toyield the coding sequence

Atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccgagcgcaaagtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtatctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaggaggagga // (SEQ ID NO:24), encoding the amino acid sequencemewswvflifisvttgvhserkvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwingkeykckvsnkglpapiektisktkgqprepqvyfippsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggg// (SEQ ID NO:25).

The pTT14-hIgG2-Fc vector with the ShK removed was treated with CalfIntestine Phosphatase (CIP) to remove the 5′ Phosphate group andPhenol/Chloroform extracted to prevent religation of the vector uponitself. The insert ShK[1-35, Q16K] fragment was gel purified away fromits vector and cleaned up with Qiagen Gel Purification Kit. The purifiedinsert was ligated to the large vector fragment and transformed intoOneShot Top10 bacteria. DNAs from transformed bacterial colonies wereisolated and subjected to BamHI restriction enzyme digestion andresolved on a one percent agarose gel. DNAs resulting in an expectedpattern were submitted for sequencing. Although, analysis of severalsequences of clones yielded a 100% percent match with the abovesequence, only one clone was selected for large scaled plasmidpurification. The final pTT14-IgG2-Fc-ShK[1-35, Q16K] construct encodedthe following IgG2 Fc-L10-ShK(1-35, Q16K) fusion protein sequence:

(SEQ ID NO: 26) mewswvflfflsvttgvhserkvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsrscidtipksrctafkckhsmkyrlsfcrktcgtc//.

The amino acid sequence for IgG2 Fc-L10-ShK(1-35) is:

(SEQ ID NO: 30) mewswvflfflsvttgvhserkvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsrscidtipksrctafqckhsmkyrlsfcrktcgtC//.

The desired aKLH IgG2/Fc-ShK product contained one copy of each ofcomponents (a)-(c), immediately above, configured as in FIG. 1E. Becauseof this, the ratio was 1:1:1. This product can be described as halfantibody and half Fc fusion (“hemibody”), coupled together at the Fcdomain. Additional peptide assemblies that had to be removed from theculture were the aKLH Ab and the Fc-ShK homodimer.

The ShK[1-35]WT fragment was generated using the originalFc-L10-ShK[1-35] in pcDNA3.1(+)CMVi as a template (described in Example1, FIGS. 14A-14B in Sullivan et al., Toxin Peptide Therapeutic Agents,PCT/US2007/022831, published as WO 2008/088422, which is incorporatedherein by reference in its entirety) and the oligos:

(SEQ ID NO: 47) 5′-TCC CTG TCT CCG GGT GGA GGA GGA GGA TCC GGA G-3′; and(SEQ ID NO: 14) 5′-CAT GCG GCC GCT CAT TAG CAG GTG-3′.

The PCR products were run on a 1% agarose gel. The bands were punchedfor an agarose plug and the plugs were placed in a fresh PCR reactiontube. The agarose plugs were then amplified by PCR using the outsideprimers SEQ ID NO:357 and SEQ ID NO:330. The PCR product was thendigested by XbaI and NotI and PCR clean up kit (Qiagen) purified. At thesame time, pTT5 Vector (an Amgen vector containing a CMV promoter andPoly A tail) was cut by XbaI and NotI. The pTT5 vector was run out on a1% agarose gel and the larger fragment was cut out and gel purified byQiagen's Gel Purification Kit. The purified PCR product was ligated tothe large vector fragment and transformed into OneShot Top10 bacteria.DNAs from transformed bacterial colonies were isolated and subjected toXbaI and NotI restriction enzyme digestions and resolved on a onepercent agarose gel. DNAs resulting in an expected pattern weresubmitted for sequencing. Although, analysis of several sequences ofclones yielded a 100% percent match with the above sequence, only oneclone was selected for large scaled plasmid purification. The finalpTT5-aKLH 120.6-VK1SP-IgG2-HC-L10-ShK[1-35] construct encoded anIgG2-HC-L10-ShK[1-35] fusion polypeptide with the amino acid sequence:

(SEQ ID NO: 48) Mdmrvpaqllgllllwlrgarcqvqlvqsgaevkkpgasvkvsckasgytftgyhmhwvrqapgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistaymelsrlrsddtavyycardrgsyywfdpwgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgggggsggggsrscidtipksrctafqckhsmkyr lsfcrktcgtc//.

To generate the ShK[1-35, Q16K] mutant version of this construct,site-directed mutagenesis was performed using the Stratagene QuikchangeMulti site Directed Mutagenesis Kit (Cat#200531), per manufacturer'sinstructions, and oligos:

5′-GCT GCA CCG CCT TCA AGT GCA AGC ACA GC 3′ (SEQ ID NO:9); and

5′-GCT GTG CTT GCA CTT GAA GGC GGT GCA GC-3′ (SEQ ID NO:10). The finalconstruct pTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK[1-35, Q16K] encodedIgG2-HC-L10-ShK[1-35, Q16K] fusion polypeptide with the following aminoacid sequence:

(SEQ ID NO: 49) Mdmrvpaqllgllllwlrgarcqvqlvqsgaevkkpgasvkvsckasgytftgyhmhwvrqapgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistaymelsrlrsddtavyycardrgsyywfdpwgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgggggsggggsrscidtipksrctafkckhsmkyr lsfcrktcgtc//.

aKLH-IgG2 Heavy Chain-L10-ShK[2-35, Q16K] Mammalian Expression.

Using DNA construct pTT5-aKLH 120.6-VK1SP-IgG2-HC-L10-ShK[1-35] as thevector, the ShK[1-35] was cut out using BamHI/BamHI. The vector fragmentfrom pTT5-aKLH 120.6-VK1SP-IgG2-HC without ShK[1-35] contained thecoding sequence:

(SEQ ID NO: 50) atggacatgagggtgcccgctcagctcctggggctcctgctgctgtggctgagaggtgccagatgtcaggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctaccacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtgcgagagatcgtgggagctactactggttcgacccctggggccagggaaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgg aggagga//,

encoding the amino acid sequence

(SEQ ID NO: 51) mdmrvpaqllgllllwlrgarcqvqlvqsgaevkkpgasvkvsckasgytftgyhmhwvrqapgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistaymelsrlrsddtavyycardrgsyywfdpwgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgggg//.

The vector fragment was then treated with Calf Intestine Phosphatase(CIP) to remove the 5′ Phosphate group and Phenol/Chloroform extractedto prevent religation of the vector upon itself. The insert came frompTT14-VH21SP-IgG2-Fc-ShK[2-35, Q16K] encoding IgG2 Fc-L10-ShK(2-35,Q16K):

(SEQ ID NO: 18) mewswvflfflsvttgvhserkvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykekvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsscidtipksrctafkckhsmkyrlsfcrktcgtc//,

and the insert was also digested out using BamHI/BamHI. The insertShK[2-35, Q16K] fragment was gel purified away from its vector andcleaned up with Qiagen Gel Purification Kit. A purified DNA insertcontaining the coding sequence

(SEQ ID NO: 52) gga tcc gga gga gga gga agc agc tgc atcgac acc atc ccc aag agc cgc tgc acc gccttc aag tgc aag cac agc atg aag tac cgcctg agc ttc tgc cgc aag acc tgc ggc acc tgc taa tga//,

encoding the amino acid sequence

(SEQ ID NO: 53) GSGGGGSSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC,was ligated to the large vector fragment and transformed into OneShotTop10 bacteria. DNAs from transformed bacterial colonies were isolatedand subjected to BamHI restriction enzyme digestion and resolved on aone percent agarose gel. DNAs resulting in an expected pattern weresubmitted for sequencing. Although, analysis of several sequences ofclones yielded a 100% percent match with the above sequence, only oneclone was selected for large scaled plasmid purification. The finalconstruct pTT5-aKLH-IgG2 HC-L10-ShK[2-35,Q16K] encoded an IgG2HC-L10-ShK[2-35,Q16K] fusion polypeptide:

(SEQ ID NO: 54) Mdmrvpaqllgllllwlrgarcqvqlvqsgaevkkpgasvkvsckasgytftgyhmhwvrqapgqglewmgwinpnsggtnyaqkfqgrytmtrdtsistaymelsrlrsddtavyycardrgsyywfdpwgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgggggsggggsscidtipksrctafkckhsmkyrl sfcrktcgtc//.

VH21SP-N-Terminus ShK[1-35] Wild Type-IgG1-Fc Mammalian Expression.

A DNA sequence coding for a monomer of the Kv1 3 inhibitor peptideShK[1-35] fused in-frame to the N-terminal Fc region of human IgG1 wasconstructed as described below.

For construction of VH21 SP-ShK(1-35)-L10-IgG1 Fc expression vector, aPCR strategy was employed to generate the VH21 signal peptide ShK(1-35)gene linked to a four glycine and one serine amino acid flanked byHindIII and BamHI restriction sites and a four glycine and one serineamino acid linked to IgG1 Fc fragment flanked by BamHI and NotIrestriction sites was generated in a PCR reaction using theFc-L10-OSKlin pcDNA3.1(+)CMVi as a template (described in Example 41 andFIGS. 42A-B of Sullivan et al., WO 2008/088422A2, incorporated byreference).

To generate VH21 SP-ShK(1-35)-G₄S, two oligos with the sequence asdepicted below were used in a PCR reaction with PfuTurbo HotStart DNApolymerase (Stratagene) at 95° C.-30 sec, 55° C.-30 sec, 75° C.-45 secfor 35 cycles; HindIII (aagctt) and BamHI (ggatcc) restriction sites areunderlined:

Forward primer: (SEQ ID NO: 55)tgcagaagcttctagaccaccatggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcccgcagctgcatcgacaccatccccaagagccgctgcaccgccttccagt//; and Reverse primer: (SEQ ID NO: 56)Ctccggatcctcctcctccgcaggtgccgcaggtcttgcggcagaagctcaggcggtacttcatgctgtgcttgcactggaaggcggtgcagcggctctt ggggatggtgtcgat//.

The resulting PCR products were resolved as the 202 bp bands on a twopercent agarose gel. The 202 bp PCR product was purified using PCRPurification Kit (Qiagen), then digested with HindIII and BamHI (Roche)restriction enzymes, and agarose gel was purified by Gel Extraction Kit(Qiagen).

To generate G₄S-IgG1 Fc, two oligos with the sequence as depicted belowwere used in a PCR reaction with PfuTurbo HotStart DNA polymerase(Stratagene) at 95° C.-30 sec, 55° C.-30 sec, 75° C.-1 min for 30cycles; BamHI (ggatcc) and NotI (gcggccgc) restriction sites areunderlined:

Forward primer: (SEQ ID NO: 57)gtaggatccggaggaggaggaagcgacaaaactcacac//; and Reverse primer:(SEQ ID NO: 58) Cgagcggccgcttactatttacccggagacaggga//.

The resulting PCR products were resolved as the 721-bp bands on a onepercent agarose gel. The 721-bp PCR product was purified using PCRPurification Kit (Qiagen), then digested with BamHI and NotI (Roche)restriction enzymes, and agarose gel was purified by Gel Extraction Kit(Qiagen).

The pcDNA3.1(+)CMVi-Fc-L10-OSK1 vector was digested with BamHI and NotIrestriction enzymes and the large fragment was purified by GelExtraction Kit. The gel purified 4GS-IgG1 Fc fragment was ligated to thepurified large fragment and transformed into One Shot® Top10(Invitrogen) to create a pCMVi-Fc-L10-IgG1 Fc vector. Subsequently,pCMVi-Fc-L10-IgG1 Fc vector was digested with HindIII and BamHIrestriction enzymes and the large fragment was purified by GelExtraction Kit. The gel purified VH21 SP-ShK(1-35)-4GS fragment wasligated to the purified large fragment and transformed into One Shot®Top10 (Invitrogen) resulting in a pCMVi-VH21 SP-ShK(1-35)-L10-IgG1 Fcconstruct. DNAs from transformed bacterial colonies were isolated anddigested with BamHI and NotI restriction enzymes and resolved on a onepercent agarose gel. DNAs resulting in an expected pattern weresubmitted for sequencing. Although, analysis of several sequences ofclones yielded a 100% percent match with the above sequences, only oneclone from each gene was selected for large scaled plasmid purification.The DNA from VH21 SP-ShK(1-35)-L10-IgG1 Fc in pCMVi vector wasresequenced to confirm the Fc and linker regions and the sequence was100% identical to the above sequence. Fragment VH21SP-ShK(1-35)-L10-IgG1 Fc contained the coding sequence

(SEQ ID NO: 59) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcccgcagctgcatcgacaccatccccaagagccgctgcaccgccttccagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgcggaggaggaggatccggaggaggaggaagcgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatagtaa//,

encoding VH21 SP-ShK(1-35)-L10-IgG1 Fc amino acid sequence

(SEQ ID NO: 60) mewswvflfflsvttgvhsrscidtipksrctafqckhsmkyrlsfcrktcgtcggggsggggsdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwpidgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk//.

Mammalian Expression of N-Terminus ShK[1-35, Q16K]-aKLH HC; andN-Terminus ShK[1-35Q16K]-aKLH LC.

Using a construct encoding N-terminus ShK[1-35]Wild Type-L10-IgG1-Fc,site directed mutagenesis was performed using the following oligos toproduce a Q16K mutation in the ShK region:

(SEQ ID NO: 9) 5′-GCT GCA CCG CCT TCA AGT GCA AGC ACA GC-3′//; and(SEQ ID NO: 10) 5′-GCT GTG CTT GCA CTT GAA GGC GGT GCA GC-3′.

The Stratagene QuikChange Multi Site Directed Mutagenesis Kit was usedaccording to the manufacturer's instructions. The final construct forpCMVi-N-terminus-ShK[1-35 Q16K]-L10-IgG1-Fcencoded the following Signalpeptide (VH21 SP)-ShK[1-35, Q16K]-L10-IgG1-Fc fusion polypeptide:

(SEQ ID NO: 61) Mewswvflfflsvttgvhsrscidtipksrctafkckhsmkyrlsfcrktcgtcggggsggggsdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk//.

To generate the N-terminus ShK[1-35, Q16K]-aKLH HC construct, a PCRproduct containing the Signal peptide-ShK[1-35Q16K]-L10 linker wasproduced using the following oligos:

(SEQ ID NO: 62) 5′-CAT TCT AGA CCA CCA TGG AAT GG-3′; (SEQ ID NO: 63)5′-CAG CTG CAC CTG GCT TCC TCC TCC TCC GG-3′;

and template pCMVi-N-terminus-ShK[1-35, Q16K]-L10-IgG1-Fc, resulted in afragment containing the coding sequence

(SEQ ID NO: 64) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcccgcagctgcatcgacaccatccccaagagccgctgcaccgccttcaagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgcggaggaggaggatccggaggaggaggaagc//,

encoding the VH21 SP-ShK(1-35, Q16K)-L10 amino acid sequence

(SEQ ID NO: 65) MEWSWVFLFFLSVTTGVHSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTCGGGGSGGGGS//.

To generate the aKLH-HC fragment, a PCR product was created usingoligos:

(SEQ ID NO: 66) 5′-GGA GGA GGA AGC CAG GTG CAG CTG GTG CAG-3′;(SEQ ID NO: 67) 5′-CAT GCG GCC GCT CAT TTA CCC-3′;

and template pTT5-aKLH 120.6-HC, resulting in a DNA fragment containingthe coding sequence

(SEQ ID NO: 68) caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctaccacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtgcgagagatcgtgggagctactactggttcgacccctggggccagggaaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga//,

encoding amino acid sequence

(SEQ ID NO: 69) qvqlvqsgaevkkpgasvkvsckasgytftgyhmhwvrqapgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistaymelsrlrsddtavyycardrgsyywfdpwgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk//.

The two PCR products were run out on a gel and the appropriate sizedband was punched for an agarose plug. The agarose plugs were placed in asingle new PCR reaction, and the fragments were sewn together usingouter most primers (SEQ ID NO:62) and (SEQ ID NO:67). The PCR fragmentwas cut using XbaI and NotI and cleaned with Qiagen PCR Cleanup Kit. Atthe same time, pTT5 vector was also cut by XbaI and NotI and gelpurified. The purified insert was ligated to the large vector fragmentand transformed into OneShot Top10 bacteria. DNAs from transformedbacterial colonies were isolated and subjected to XbaI and NotIrestriction enzyme digestions and resolved on a one percent agarose gel.DNAs resulting in an expected pattern were submitted for sequencing.Although, analysis of several sequences of clones yielded a 100% percentmatch with the above sequence, only one clone was selected for largescaled plasmid purification. The final construct pTT5-N-terminusShK[1-35Q16K]-L10-aKLH120.6-HC encoded a VH21 SP-ShK[1-35,Q16K]-L10-aKLH120.6-HC fusion polypeptide:

(SEQ ID NO: 70) Mewswvflfflsvttgvhsrscidtipksrctafkckhsmkyrlsfcrktcgtcggggsggggsqvqlvqsgaevkkpgasvkvsckasgytftgyhmhwvrqapgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistaymelsrlrsddtavyycardrgsyywfdpwgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqthwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytq kslslspgk//.

Lastly, the N-terminus-ShK[1-35, Q16K]-L10-aKLH120.6 Light Chain (LC)was generated in the same manner as above. A PCR product containing thesignal peptide-ShK[1-35, Q16K]-L10 was created using oligos:

(SEQ ID NO: 62) 5′-CAT TCT AGA CCA CCA TGG AAT GG-3′; and(SEQ ID NO: 71) 5′-CAT CTG GAT GTC GCT TCC TCC TCC TCC GG-3′;

and template pCMVi-N-terminus-ShK[1-35Q16K]-L10-IgG1-Fc, resulting in aDNA fragment containing the coding sequence

(SEQ ID NO: 64) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcccgcagctgcatcgacaccatccccaagagccgctgcaccgccttcaagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgcggaggaggaggatccggaggaggaggaagc//,

encoding the amino acid sequence for a signal peptide (VH21SP)-ShK(1-35, Q16K)-L10 linker

(SEQ ID NO: 65) mewswvflfflsvttgvhsrscidtipksrctafkckhsmkyrlsfcrktcgtcggggsggggs//.

Using template and oligos:

(SEQ ID NO: 72) 5′-GGA GGA GGA AGC GAC ATC CAG ATG ACC  CAG TC-3′; and(SEQ ID NO: 73) 5′-CAT CTC GAG CGG CCG CTC AAC-3′.

The resulting cloned PCR fragment contained the coding sequenceatggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcccgcagctgcatcgacaccatccccaagagccgctgcaccgccttcaagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgcggaggaggaggatccggaggaggaggaagcgacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaaccagggaaagcccctaaacgcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcctgaagattttgcaacttattactgtctacagcataatagttacccgctcactttcggcggagggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttga// (SEQ ID NO:74) was generated,

encoding the amino acid sequence for N-terminus VH21 SP-ShK[1-35,Q16KL10-aKLH120.6 Light Chain (LC) with an N-terminal signal peptide:

(SEQ ID NO: 75) mewswvflfflsvttgvhsrscidtipksrctafkckhsmkyrlsfcrktcgtcggggsggggsdiqmtqspsslsasvgdrvtitcrasqgirndlgwyqqkpgkapkrliyaasslqsgvpsrfsgsgsgteftltisslqpedfatyyclqhnsypltfgggtkveikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec//.

Both PCR fragments (DNA fragment containing the coding sequence (SEQ IDNO:64) and aKLH 120.6 Light Chain LC fragment containing the codingsequence (SEQ ID NO:74) were run out on a gel, and the appropriate sizedband was punched for an agarose plug. The agarose plugs were placed in asingle new PCR reaction, and the fragments were sewn together usingouter most primers (SEQ ID NO:62) and (SEQ ID NO:73). The resulting PCRfragment was cut using XbaI and NotI and cleaned with Qiagen PCR CleanupKit.

At the same time, pTT14 vector (an Amgen vector containing a CMVpromoter, Poly A tail and a Puromycin resistance gene) was also cut byXbaI and NotI and gel purified. The purified insert was ligated to thelarge vector fragment and transformed into OneShot Top10 bacteria. DNAsfrom transformed bacterial colonies were isolated and subjected to XbaIand NotI restriction enzyme digestions and resolved on a one percentagarose gel. DNAs resulting in an expected pattern were submitted forsequencing. The final construct pTT14-N-terminusShK[1-35Q16K]-L10-aKLH120.6-LC encoding a Signal Peptide-ShK[1-35,Q16K]-L10-aKLH120.6-LC fusion polypeptide sequence (i.e., SEQ ID NO:75).

Example 9 Purifications and Evaluation of Comparator MoleculesMonovalent Fc/Fc-L10-ShK[2-35] Heterodimers and Monovalent or BivalentFc/Fc-ShK(1-35 Q16K)(IgG2) Heterodimers and Other Polypeptide Molecules

Monovalent or bivalent Fc-L10-ShK[2-35], monovalent or bivalentFc-L10-ShK[1-35], monovalent or bivalent Fc-L10-ShK(1-35, Q16K), andother ShK-related polypeptide molecules listed in Table 7B (in Example 5herein), were expressed, isolated and purified by methods describedherein. PEGylated and un-PEGylated toxin peptide comparators in Table 7Bwere prepared synthetically as follows:

Peptide Synthesis.

N′-Fmoc, side-chain protected amino acids and H-Cys(Trt)-2Cl-Trt resinwere purchased from Novabiochem, Bachem, or Sigma Aldrich. The followingside-chain protection strategy was employed: Asp(OtBu), Arg(Pbf),Cys(Trt), Glu(OtBu), His(Trt), Lys(N^(ε)-Boc), Ser(OtBu), Thr(OtBu) andTyr(OtBu). ShK (RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC// SEQ ID NO:378),[Lys16]ShK (RSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC// SEQ ID NO:76), orother toxin peptide analog amino acid sequences, were synthesized in astepwise manner on an CS Bio peptide synthesizer by SPPS using DIC/HOBtcoupling chemistry at 0.2 mmol equivalent scale using H-Cys(Trt)-2Cl-Trtresin (0.2 mmol, 0.32 mmol/g loading). For each coupling cycle, 1 mmolN^(α)-Fmoc-amino acid was dissolved in 2.5 mL of 0.4 M1-hydroxybenzotriazole (HOBt) in N,N-dimethylformamide (DMF). To thesolution was added 1.0 mL of 1.0 M N,N′-diisopropylcarbodiimide (DIC) inDMF. The solution was agitated with nitrogen bubbling for 15 min toaccomplish pre-activation and then added to the resin. The mixture wasshaken for 2 h. The resin was

filtered and washed three times with DMF, twice with dichloromethane(DCM), and three times with DMF. Fmoc deprotections were carried out bytreatment with 20% piperdine in DMF (5 mL, 2×15 min). The first 23residues were single coupled through repetition of the Fmoc-amino acidcoupling and Fmoc removal steps described above. The remaining residueswere double coupled by performing the coupling step twice beforeproceeding with Fmoc-removal.

Following synthesis, the resin was then drained, and washed sequentiallywith DCM, DMF, DCM, and then dried in vacuo. The peptide-resin wastransferred to a 250-mL plastic round bottom flask. The peptide wasdeprotected and released from the resin by treatment withtriisopropylsilane (1.5 mL), 3,6-dioxa-1,8-octane-dithiol (DODT, 1.5mL), water (1.5 mL), trifluoroacetic acid (TFA, 20 mL), and a stir bar,and the mixture was stirred for 3 h. The mixture was filtered through a150-mL sintered glass funnel into a 250-mL plastic round bottom flask.The mixture was filtered through a 150-mL sintered glass funnel into a250-mL plastic round bottom flask, and the filtrate was concentrated invacuo. The crude peptide was precipitated with the addition of colddiethyl ether, collected by centrifugation, and dried under vacuum.

Peptide Folding. The dry crude linear peptide (about 600 mg), forexample [Lys16]ShK peptide (SEQ ID NO:76) or [Lys16]ShK-Ala (also knownas [Lys16, A1a36]-ShK; SEQ ID NO:379) peptide, was dissolved in 16 mLacetic acid, 64 mL water, and 40 mL acetonitrile. The mixture wasstirred rapidly for 15 min to complete dissolution. The peptide solutionwas added to a 2-L plastic bottle that contained 1700 mL of water and alarge stir bar. To the thus diluted solution was added 20 mL ofconcentrated ammonium hydroxide to raise the pH of the solution to 9.5.The pH was adjusted with small amounts of acetic acid or NH₄OH asnecessary. The solution was stirred at 80 rpm overnight and monitored byLC-MS. Folding was usually judged to be complete in 24 to 48 h, and thesolution was quenched by the addition of acetic acid and TFA (pH=2.5).The aqueous solution was filtered (0.45 μm cellulose membrane).

Reversed-Phase HPLC Purification.

Reversed-phase high-performance liquid chromatography was performed onan analytical (C18, 5 μm, 0.46 cm×25 cm) or a preparative (C18, 10 μm,2.2 cm×25 cm) column. Chromatographic separations were achieved usinglinear gradients of buffer B in A (A=0.1% aqueous TFA; B=90% aq. ACNcontaining 0.09% TFA) typically 5-95% over 35 min at a flow rate of 1mL/min for analytical analysis and 5-65% over 90 min at 20 mL/min forpreparative separations. Analytical and preparative HPLC fractions werecharacterized by ESMS and photodiode array (PDA) HPLC, combined andlyophilized.

Mass Spectrometry.

Mass spectra were acquired on a single quadrupole mass spectrometerequipped with an Ionspray atmospheric pressure ionization source.Samples (25 μL) were injected into a moving solvent (10 μL/min; 30:50:20ACN/MeOH containing 0.05% TFA) coupled directly to the ionization sourcevia a fused silica capillary interface (50 μm i.d.). Sample dropletswere ionized at a positive potential of 5 kV and entered the analyzerthrough an interface plate and subsequently through an orifice (100-120μm diameter) at a potential of 60 V. Full scan mass spectra wereacquired over the mass range 400-2200 Da with a scan step size of 0.1Da. Molecular masses were derived from the observed m/z values.

PEGylation, Purification and Analysis.

Peptide, e.g., [Lys16]ShK (SEQ ID NO:76) or [Lys16]ShK-Ala (SEQ IDNO:379), was selectively PEGylated by reductive alkylation at itsN-terminus, using activated linear or branched PEG. Conjugation wasperformed at 2 mg/ml in 50 mM NaH₂PO₄, pH 4.5 reaction buffer containing20 mM sodium cyanoborohydride and a 2 molar excess of 20 kDamonomethoxy-PEG-aldehyde (NOF, Japan). Conjugation reactions werestirred for approximately 5 hrs at room temperature, and their progresswas monitored by RP-HPLC. Completed reactions were quenched by 4-folddilution with 20 mM NaOAc, pH 4 and chilled to 4° C. The PEG-peptideswere then purified chromatographically at 40 C; using SP Sepharose HPcolumns (GE Healthcare, Piscataway, N.J.) eluted with linear 0-1M NaClgradients in 20 mM NaOAc, pH 4.0. Eluted peak fractions were analyzed bySDS-PAGE and RP-HPLC and pooling determined by purity >97%. Principlecontaminants observed were di-PEGylated toxin peptide analog. Selectedpools were concentrated to 2-5 mg/ml by centrifugal filtration against 3kDa MWCO membranes and dialyzed into 10 mM NaOAc, pH 4 with 5% sorbitol.Dialyzed pools were then sterile filtered through 0.2 micron filters andpurity determined to be >97% by SDS-PAGE (data not shown). Reverse-phaseHPLC was performed on an Agilent 1100 model HPLC running a Zorbax® 5 μm300SB-C8 4.6×50 mm column (Agilent) in 0.1% TFA/H₂O at 1 ml/min andcolumn temperature maintained at 40° C. Samples of PEG-peptide (20 μg)were injected and eluted in a linear 6-60% gradient while monitoringwavelength 215 nm.

Fusion Proteins.

Generally, FIG. 1A and FIG. 1B show a schematic representation ofmonovalent and bivalent Fc-toxin peptide (or toxin peptide analog)fusion proteins (or “peptibodies”), respectively. The bivalent Fc-ShKmolecule is a homodimer containing two Fc-ShK chains. The monovalentFc-ShK toxin peptide (or toxin peptide analog) molecule is a heterodimercontaining one Fc chain and one Fc-ShK (or analog) chain. Since themonovalent Fc-ShK molecule contains just a single ShK peptide per dimer,it is considered monovalent. Constructs or chains referred to asFc-(toxin peptide analog), contain an N-terminal Fc region and anoptional flexible linker sequence (e.g., L10 peptidyl linker GGGGSGGGGS;SEQ ID NO:153) covalently attached to the toxin peptide or toxin peptideanalog, such that the orientation from N- to C-terminus would be:Fc-linker-toxin peptide or toxin peptide analog.

In Examples 1 and 2 of Sullivan et al., WO 2008/088422A2, were describedthe activity of bivalent Fc-ShK peptibodies, Fc-L10-ShK(1-35) andFc-L10-ShK(2-35) expressed from mammalian cells. In Example 1 of WO2008/088422A2, was also described isolation of a monovalentFc-L10-ShK(1-35) molecule, formed as a small by-product duringexpression. The monovalent antibody #3742-ShK(1-35, Q16K) conjugateprovided potent blockade of T cell cytokine secretion in human wholeblood (see, Table 7A-B, in Example 5 herein).

Example 10 Pharmacokinetic (PK) Studies in Rats and Cynomolgus Monkeys

Rat PK.

The pharmacokinetic profiles of the 16435 and 4341 antibodies weredetermined in adult Sprague-Dawley (SD) rats (n=3 per group) byinjecting 5 mg/kg subcutaneously and collecting approximately 250 μL ofblood in Microtainer® serum separator tubes at 0, 0.25, 1, 4, 24, 48,72, 168, 336, 504, 672, 840 and 1008 hours post-dose from the lateraltail vein. Each sample was maintained at room temperature followingcollection, and following a 30-40 minute clotting period, samples werecentrifuged at 2-8° C. at 11,500 rpm for about 10 minutes using acalibrated Eppendorf 5417R Centrifuge System (Brinkmann Instruments,Inc., Westbury, N.Y.). The collected serum was then transferred into apre-labeled (for each rat), cryogenic storage tube and stored at −60° C.to −80° C. for future analysis. To measure the serum sampleconcentrations from the PK study samples, the following method was used:½ area black plate (Corning 3694) was coated with 2 μg/ml of anti-hu Fc,antibody 1.35.1 in PBS and then incubated overnight at 4° C. The platewas then washed and blocked with I-Block™ (Applied Biosystems) overnightat 4° C. If samples needed to be diluted, then they were diluted in RatSD serum. The standards and samples were then diluted 1:20 in 1×PBS+1MNaCl+0.5% Tween 20 and 1% BSA buffer (5% serum). The plate was washedand 50-μl samples of diluted standards and samples were transferred intoan antibody 1.35.1 coated plate and incubated for 1.5 h at roomtemperature. The plate was washed, then 50 μl of 100 ng/ml of anti-hu Fcantibody 21.1-HRP conjugate in I-Block™+5% BSA was added and incubatedfor 1.5 h. The plate was washed, then 50 μl of Pico substrate wereadded, after which the plate was immediately analyzed with aluminometer. Time concentration data were analyzed usingnon-compartmental methods with WinNonLin® (Enterprise version 5.1.1,2006, Pharsight® Corp. Mountain View, Calif.) (FIG. 34.0). Thepharmacokentic profiles of these two antibodies in Sprague-Dawley ratare shown in FIG. 43. The PK parameters of 16435 and 4341 antibodies inSD Rats are summarized in the Table 8 (below). Both molecules have goodPK profile in rats with half life of over 10 days.

TABLE 8 PK parameters of antibodies 16435 and 4341 in SD Rats. SC DoseT_(1/2) Tmax Cmax MRT CL/F AUC_(0-t) AUC_(0-inf) Compound (mg/kg) (h)(h) (ng/mL) (h) (mL/h/kg) (ng · h/mL) (ng · h/mL) 16435 5 226 104 44,080395 0.368 16,038,601 20,048,353 4341 5 365 136 38,963 580 0.19022,280,335 26,661,802

Cynomolgus PK.

The pharmacokinetic profiles of the 16435 and 4341 antibodies were alsodetermined in cynomolgus monkeys (n=2 per group) by injecting of twosubsequent subcutaneous doses of 1 mg/kg at day 0 and 5 mg/kg at day 57.Serum samples were collected at pre-dose, 0.5, 2, 4, 8, 12, 24, 48, 96,168, 336, 504, 672, 840, 1008, 1176, 1344 (prior to second dose) hourspost 1^(st) dose at 1 mg/kg and 0.5, 2, 4, 8, 12, 24, 48, 96, 168, 336,360, 384, 432, 504, 672, 840, 1008, 1176, 1344 following post 2^(nd)dose at 5 mg/kg. The samples were assayed for the 16435 and 4341antibody levels by using an anti-IgG sandwich ELISA as described above.Time concentration data were analyzed using non-compartmental methodswith WinNonLin®. The pharmacokentic profiles of these two antibodies incynomolgus monkey are shown in FIG. 44. The PK parameters of 16435 and4341 antibodies in cynomolgus monkeys are summarized in the Table 9(below). Both molecules exhibited a good PK profile in cynos, with halflife of about 12 and 21 days for 16435 and 4341, respectively. The 4341antibody has better PK attributes than 16435 and has shown normal hu IgGclearance in monkey based on FcRn binding and in the absence of anytarget mediated drug disposition (TMDD) clearance mechanism. Inaddition, the results in FIG. 44 show that even with multiple dosing inthe cynos, both antibodies 16435 and 4341 had no indication of asignficant change in the clearance mediated by an immune response in thecynos. If there had been a significant immune response causing abnormalantibody clearance, it would have been expected after the second dose,due to immune system priming by the first dose.

TABLE 9 PK parameters of antibodies 16435 and 4341 in cynomolgusmonkeys. SC Dose T_(1/2) Tmax Cmax MRT CL/F AUC_(0-t) AUC_(0-inf)Compound (mg/kg) (h) (h) (ng/mL) (h) (mL/h/kg) (ng · h/mL) (ng · h/mL)16435 5 285 96 58,682 450 0.161 29,924,604 31,230,285 4341 5 502 9668,166 740 0.096 43,578,088 51,909,826

ABBREVIATIONS

Abbreviations used throughout this specification are as defined below,unless otherwise defined in specific circumstances.

-   Ac acetyl (used to refer to acetylated residues)-   AcBpa acetylated p-benzoyl-L-phenylalanine-   ACN acetonitrile-   AcOH acetic acid-   ADCC antibody-dependent cellular cytotoxicity-   Aib aminoisobutyric acid-   bA beta-alanine-   Bpa p-benzoyl-L-phenylalanine-   BrAc bromoacetyl (BrCH₂C(O)-   BSA Bovine serum albumin-   Bzl Benzyl-   Cap Caproic acid-   CBC complete blood count-   COPD Chronic obstructive pulmonary disease-   CTL Cytotoxic T lymphocytes-   DCC Dicylcohexylcarbodiimide-   Dde 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl-   DNP 2,4-dinitrophenol-   DOPC 1,2-Dioleoyl-sn-Glycero-3-phosphocholine-   DOPE 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine-   DPPC 1,2-Dipalmitoyl-sn-Glycero-3-phosphocholine-   DSPC 1,2-Distearoyl-sn-Glycero-3-phosphocholine-   DTT Dithiothreitol-   EAE experimental autoimmune encephalomyelitis-   ECL enhanced chemiluminescence-   ESI-MS Electron spray ionization mass spectrometry-   FACS fluorescence-activated cell sorting-   Fmoc fluorenylmethoxycarbonyl-   GHT glycine, hypoxanthine, thymidine-   HOBt 1-Hydroxybenzotriazole-   HPLC high performance liquid chromatography-   HSL homoserine lactone-   IB inclusion bodies-   KCa calcium-activated potassium channel (including IKCa, BKCa, SKCa)-   KLH Keyhole Limpet Hemocyanin-   Kv voltage-gated potassium channel-   Lau Laurie acid-   LPS lipopolysaccharide-   LYMPH lymphocytes-   MALDI-MS Matrix-assisted laser desorption ionization mass    spectrometry-   Me methyl-   MeO methoxy-   MeOH methanol-   MHC major histocompatibility complex-   MMP matrix metalloproteinase-   MW Molecular Weight-   MWCO Molecular Weight Cut Off-   1-Nap 1-napthylalanine-   NEUT neutrophils-   Nle norleucine-   NMP N-methyl-2-pyrrolidinone-   OAc acetate-   PAGE polyacrylamide gel electrophoresis-   PBMC peripheral blood mononuclear cell-   PBS Phosphate-buffered saline-   Pbf 2,2,4,6,7-pendamethyldihydrobenzofuran-5-sulfonyl-   PCR polymerase chain reaction-   PD pharmacodynamic-   Pec pipecolic acid-   PEG Poly(ethylene glycol)-   pGlu pyroglutamic acid-   Pic picolinic acid-   PK pharmacokinetic-   pY phosphotyrosine-   RBS ribosome binding site-   RT room temperature (about 25° C.)-   Sar sarcosine-   SDS sodium dodecyl sulfate-   STK serine-threonine kinases-   t-Boc tert-Butoxycarbonyl-   tBu tert-Butyl-   TCR T cell receptor-   TFA trifluoroacetic acid-   THF thymic humoral factor-   Trt trityl

1. An isolated immunoglobulin, comprising an immunoglobulin heavy chainvariable region and an immunoglobulin light chain variable region,wherein the light chain variable region comprises an amino acid sequencethat is at least 95% identical to the amino acid sequence of SEQ IDNO:202 and the heavy chain variable region comprises an amino acidsequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO:349.
 2. The isolated immunoglobulin of claim 1, wherein: (a)the light chain variable region comprises the amino acid sequence of SEQID NO:196 and the heavy chain variable region comprises the amino acidsequence of SEQ ID NO:335, SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353,SEQ ID NO:355, or SEQ ID NO:359; or (b) the light chain variable regioncomprises the amino acid sequence of SEQ ID NO:204 and the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO:349 orSEQ ID NO:355; or (c) the light chain variable region comprises theamino acid sequence of SEQ ID NO:202 and the heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO:349; or (d) the lightchain variable region comprises the amino acid sequence of SEQ ID NO:192and the heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:357, SEQ ID NO:359, or SEQ ID NO:369; or (e) the light chainvariable region comprises the amino acid sequence of SEQ ID NO:194 andthe heavy chain variable region comprises the amino acid sequence of SEQID NO:335, SEQ ID NO:349, or SEQ ID NO:351.
 3. (canceled)
 4. Theisolated immunoglobulin of claim 2, wherein the light chain variableregion comprises the amino acid sequence of SEQ ID NO:196; and the heavychain variable region comprises the amino acid sequence of SEQ IDNO:353.
 5. The isolated immunoglobulin of claim 2, wherein the lightchain variable region comprises the amino acid sequence of SEQ IDNO:202; and the heavy chain variable region comprises the amino acidsequence of SEQ ID NO:349.
 6. (canceled)
 7. The isolated immunoglobulinof claim 1, wherein the isolated immunoglobulin comprises an antibody orantibody fragment.
 8. The isolated immunoglobulin of claim 7, comprisingan IgG1, IgG2, IgG3 or IgG4.
 9. The isolated immunoglobulin of claim 7,wherein the antibody is a monoclonal antibody.
 10. The isolatedimmunoglobulin of claim 9, wherein the monoclonal antibody is a humanantibody.
 11. The isolated immunoglobulin of claim 10, comprising: (a)an immunoglobulin heavy chain comprising the amino acid sequence of SEQID NO:101, or comprising the foregoing sequence from which one, two,three, four or five amino acid residues are lacking from the N-terminusor C-terminus, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:98, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminus or C-terminus, or both; or (b) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO:119, or comprising the foregoing sequence from which one, two, three,four or five amino acid residues are lacking from the N-terminus orC-terminus, or both; and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:116, or comprising the foregoingsequence from which one, two, three, four or five amino acid residuesare lacking from the N-terminus or C-terminus, or both.
 12. The isolatedimmunoglobulin of claim 1, further comprising at least onepharmacologically active chemical moiety conjugated thereto.
 13. Theisolated immunoglobulin of claim 12, wherein the pharmacologicallyactive chemical moiety is a pharmacologically active polypeptide. 14.The isolated immunoglobulin of claim 13, wherein the immunoglobulin isrecombinantly produced.
 15. The isolated immunoglobulin of claim 14,wherein the immunoglobulin comprises at least one immunoglobulin heavychain and at least one immunoglobulin light chain, and wherein thepharmacologically active polypeptide is inserted in the primary aminoacid sequence of the of the immunoglobulin heavy chain within aninternal loop of the Fc domain of the immunoglobulin heavy chain. 16.The isolated immunoglobulin of claim 13, wherein the immunoglobulincomprises at least one immunoglobulin heavy chain and at least oneimmunoglobulin light chain, and wherein the pharmacologically activepolypeptide is conjugated at the N-terminus or C-terminus of theimmunoglobulin heavy chain.
 17. The isolated immunoglobulin of claim 13,wherein the immunoglobulin comprises at least one immunoglobulin heavychain and at least one immunoglobulin light chain, and wherein thepharmacologically active polypeptide is conjugated at the N-terminus orC-terminus of the immunoglobulin light chain.
 18. The isolatedimmunoglobulin of claim 13, wherein the pharmacologically activepolypeptide is a toxin peptide or peptide analog, an IL-6 bindingpeptide, a CGRP peptide antagonist, a bradykinin B1 receptor peptideantagonist, a PTH agonist peptide, a PTH antagonist peptide, an ang-1binding peptide, an ang-2 binding peptide, a myostatin binding peptide,an EPO-mimetic peptide, a FGF21 peptide, a TPO-mimetic peptide, a NGFbinding peptide, a BAFF antagonist peptide, a GLP-1 or peptide mimeticthereof, or a GLP-2 or peptide mimetic thereof.
 19. The isolatedimmunoglobulin of claim 18, wherein the toxin peptide or peptide analogis ShK or a ShK peptide analog.
 20. A pharmaceutical compositioncomprising the immunoglobulin of claim 1; and a pharmaceuticallyacceptable diluent, excipient or carrier. 21-36. (canceled)
 37. Theimmunoglobulin of claim 1, wherein the immunoglobulin at 10 micromolarconcentration does not significantly bind soluble human TR2 (SEQ IDNO:82) at 10 nanomolar concentration in an aqueous solution incubatedunder physiological conditions, as measured by a surface plasmonresonance binding assay.
 38. The isolated immunoglobulin of claim 13,wherein the pharmacologically active polypeptide is conjugated to theimmunoglobulin via a functional group on a side chain of an amino acidresidue within the primary sequence of the immunoglobulin.
 39. Theisolated immunoglobulin of claim 13, wherein the pharmacologicallyactive polypeptide is conjugated to the immunoglobulin via anon-peptidyl or peptidyl linker.
 40. The isolated immunoglobulin ofclaim 39, wherein the peptidyl linker comprises glycine, serine,alanine, or combinations thereof.
 41. The isolated immunoglobulin ofclaim 39, wherein the non-peptidyl linker is a polyethylene glycollinker.
 42. The isolated immunoglobulin of claim 18, wherein thepharmacologically active polypeptide is a toxin peptide or peptideanalog.